Journal articles on the topic 'Tail immersion technique'

A series of novel (R)-5-bromo-3-(N-methylpyrrolidine-2-yl-methyl)-1H (substituted)-indole (T1-T5) derivates were synthesized by electrophilic substitution at 1st position of (R)-5-bromo-3-(N-methylpyrrolidine-2-yl-methyl)-1Hindole with various halides. The starting material (R)-5-bromo-3-(N-methylpyrrolidine-2-yl-methyl)-1H-indole was synthesized from 4-bromo aniline by multistep synthesis. The synthesized compounds were characterized by IR, 1H NMR and MASS spectroscopy and newly synthesized compounds were evaluated for their analgesic activity by tail immersion technique using wistar albino mice. Among the synthesized compounds T3, T4, T5.have shown significant activity by tail immersion technique. Compound (R)- 5-bromo-1-ethyl-3-[(1-methylpyrrolidin-2-yl)methyl]-1Hindole (T3) emerged as the most potent analgesic agent and it is equipotent when compared to the reference standard diclofenac sodium. Keywords: Indole derivatives; Analgesic activity; Tail immersion technique.

The rhizome of Canna indica L. (family: Cannaceae) was extracted in methanol and two doses- 200 and 400 mg/kg body weight of the extract were employed to reveal central and peripheral analgesic, hypoglycemic and antidiarrheal properties in vivo. According to tail immersion technique, both doses significantly rose the time of latent response for 90 minutes compared to control mice group in assessing central analgesia. Acetic acid-induced writhing process demonstrated 37.29% and 64.41% inhibition of mice movement at both doses, respectively which indicated promising and statistically significant peripheral analgesic effect. On the other hand, hypoglycemic and antidiarrheal activities maintained dose and time dependent manner. Moreover, the extract of rhizome displayed statistically significant antidiarrheal activity at 400 mg/kg b.w. dose. Bangladesh Pharmaceutical Journal 25(2): 137-142, 2022 (July)

The rhizome of Canna indica L. (family: Cannaceae) was extracted in methanol and two doses- 200 and 400 mg/kg body weight of the extract were employed to reveal central and peripheral analgesic, hypoglycemic and antidiarrheal properties in vivo. According to tail immersion technique, both doses significantly rose the time of latent response for 90 minutes compared to control mice group in assessing central analgesia. Acetic acid-induced writhing process demonstrated 37.29% and 64.41% inhibition of mice movement at both doses, respectively which indicated promising and statistically significant peripheral analgesic effect. On the other hand, hypoglycemic and antidiarrheal activities maintained dose and time dependent manner. Moreover, the extract of rhizome displayed statistically significant antidiarrheal activity at 400 mg/kg b.w. dose. Bangladesh Pharmaceutical Journal 25(2): 137-142, 2022 (July)

Background. Elaeocarpus serratus L. (Family: Elaeocarpaceae) is a tropical fruit tree, traditionally used in the treatments of poisoning, diarrhea, arthritis, and other diseases. Objectives. The current study was performed to conduct the analgesic, antidiarrheal, and hypoglycemic activity of E. serratus in mice model using methanolic bark crude extract. Methods. To assess the peripheral and central analgesic activity, the acetic acid-induced writhing and tail immersion methods were respectively used. The castor-oil mediated antidiarrheal method was used to assess the antidiarrheal activity whereas, the tail tipping technique was conducted to determine the hypoglycemic activity of the plant extract. Results. In the peripheral analgesic assay, the methanolic bark crude extract of E. serratus significantly inhibits the number of writing 69.77% (200 mg/kg) and 73.26% (400 mg/kg) respectively (p<0.05) which was strongly comparable with standard NSAID drug diclofenac sodium 75.58% (p<0.05). Similarly, it shown a significant tail flicking response for 30 minutes, 60 minutes and 90 minutes of central analgesic activity assay. In antidiarrheal activity assay, the E. serratus substantially reduced the number of diarrheal feces 64.26% (200 mg/kg, p<0.05) and 78.57% (400 mg/kg, p<0.05) which was also comparable with the positive control loperamide. The hypoglycemic activity of E. serratus extract was not convincing. Conclusions. Our investigation demonstrated the significant analgesic and antidiarrheal activities of methanolic bark extract of E. serratus (200 and 400 mg/kg) in mice model.

Phytochemical analysis of Tanaecium bilabiatum leaf led to the isolation and identification of salacinin C, daturadiol and a mixture of triterpenes, α-amyrin and β-amyrin. Besides chemical analysis, the methanol extract of T. bilabiatum leaf, METBL was evaluated for analgesic, anti-diarrheal and hypoglycemic potential in mice model. For analgesic activity test by tail immersion technique, the extract significantly extended the percent elongation time of thermal nociception. METBL (400 mg/kg bw) also established the highest inhibition of formalin-induced abdominal writhing (69.39%) in mice, which was comparable with the standard aspirin (75.51%). Likewise, dose-dependent antidiarrheal activity was observed in this test. After oral administration, METBL demonstrated maximum inhibition (40.93%) of castor oil-induced diarrhea in mice. In oral glucose tolerance test, the plant extract produced significant (p < 0.05) hypoglycemic activity. Therefore, it is concluded that T. bilabiatum is a promising source of bioactive compounds with analgesic, antidiarrheal and hypoglycemic potential. Bangladesh J. Bot. 52(1): 119-127, 2023 (March)

Background: Cleome viscosa Linn a plant from the family Capparaceae has been used in traditional medicine in Asia and Africa for the treatment of various diseases including diarrhea, cardiac stimulant, anthelmintic, carminative, anti-inflammatory and antiseptic. Decoction of the whole plant has been used for the treatment of inflammation and as a stimulant while the leaves have been applied externally on wounds and ulcers. The objective of this study is to investigate and evaluate the antipyretic and analgesic properties, toxicity profile and the phytochemical study of the methanolic leaf extract of African Cleome viscosa.Methods: The antipyretic study was performed by adopting the Baker’s yeast induced pyrexia in rats while the analgesic study was performed adopting the tail immersion or tail flick technique. Data generated was analyzed as appropriate using the analysis of variance (ANOVA).Results: The methanolic extract of the leaves of the African Cleome viscosa had a significant (P<0.05) antipyretic and analgesic effects at different time intervals and varying doses when compared with the negative control groups in the respective studies. The preliminary phytochemical study showed the presence of alkaloids, tannins, steroids, glycosides and flavonoids. The extract also proved to be non-toxic in the acute toxicity study carried out at the highest dose of 5000mg/kg body weight administered to the albino rats.Conclusions: The methanolic leaf extract of the plant showed significant antipyretic and analgesic activity in albino rats thus supporting its use in traditional medicine.

Although the existence of the spermatozoon was first noted more than 300 years ago, the understanding of its function, physicochemical structure, and viability is a relatively recent discovery. The structure of the sperm is distinctive: it is composed of an oblong-shaped head, a neck, a midpiece, and a flagellating tail, and is approximately 50–75 μm long. Seminal fluid is gelatinous and sticky, and is composed of water, salts, organic matter, proteins, and lipoids in an alkaline state. Dry seminal stains appear grayish-white, and impart a stiff, starchy feeling to an absorbent material. Methods for detection of these stains include tactile and chemical tests, ultraviolet light or alternate light sources, and microcrystal tests, although noted nonspecific interferences exist for these methods. Recovery procedures of dry stains include cutting, scraping, and immersion, and acidic destruction or sonic oscillation of the substrate cloth. Unstained sperm may be microscopically detected, but may be confused with Trichomonas vaginalis or other interferences to the untrained eye. Chemical staining is utilized in order to aid in sperm identification by individually coloring the acrosome, nucleus, and/or tail. Staining, counterstaining, and other procedures for the purpose of identification are described here.

Rose is a widely favored floriculture crop that is commercially propagated through the application of tissue culture techniques. Here, we report an effective method for axillary shoot proliferation in Al-Taif rose, an important cultivar for rose oil industry. Stem nodes were excised from an adult donor Al-Taif rose shrub and cultured for 4 weeks on Murashige and Skoog’s (MS) medium supplemented with 6-benzylaminopurine (BAP) or gibberellic acid (GA3) at 0 and 3 mg·L−1 to induce the sprouting of axillary shoots. Al-Taif rose shoots were cultured in vitro for 6 weeks on MS medium fortified with different concentrations of cytokinins, light/dark incubation and different culture types (gelled and liquid/bioreactor culture). The culture conditions that were applied had a noteworthy impact on the responses of Al-Taif rose shoot proliferation. The supplementation of the medium with 6-benzylaminopurine (BAP) resulted in an augmented rate of shoot proliferation in comparison to other cytokinins. Additionally, dark incubation limited foliage growth, leaf yellowing and abscission and favored shoot proliferation compared with light incubation. Liquid culture using bioreactors provided higher axillary shoot proliferation and growth as compared with gelled culture. A continuous immersion system with a net provided the highest axillary shoots (four shoots per explant) and shoot length (16.5 cm), whereas an immersion system without a net provided the highest fresh weight of axillary shoots (499 mg per explant). These findings will improve commercial propagation and contribute to the rose oil industry of Al-Taif rose.

Abstract Scelidosaurus fossils were first discovered during the commercial quarrying of the Liassic sea-cliffs between Charmouth and Lyme Regis in Dorset during the late 1850s. The original specimens included a well-preserved skull embedded in a block of argillaceous limestone (marlstone). Shortly after this skull was retrieved, a series of more-or-less contiguous marlstone slabs were recovered, containing most of the skeleton of the same animal (NHMUK R1111). After rudimentary (hammer and chisel) mechanical preparation, Owen published descriptions of this material (Owen, 1861, 1863). These two monographs have been the sole references pertaining to the anatomy of Scelidosaurus for >150 years. The skeleton of the lectotype of Scelidosaurus harrisonii (NHMUK R1111) has since been extracted from the surrounding matrix using an acid-immersion technique. Some additional specimens held in the collections of the Natural History Museum London, the Bristol City Museum and the Sedgwick Museum in Cambridge provide anatomical material that allows detailed description of this taxon, for which we have had, until now, a surprisingly poor understanding. Axial skeleton: The axial skeleton of Scelidosaurus comprises eight cervical, 16 dorsal, four sacral and > 40 caudal vertebrae. During ontogeny, the posterior centrum articular surface of the 16th dorsal vertebra develops a firm, ligament-bonded junction with the succeeding sacral centrum. Apart from the atlas rib, which is single headed, double-headed ribs are present throughout the presacral vertebral series, and none shows any indication of fusion to its associated vertebra. However, those ribs attached to cervical vertebrae 2–4 were evidently bound firmly by connective tissue to rugose diapophyses. The last two (presacral) dorsal ribs show merger of the capitulum and tuberculum, meaning that they are separated by only a step. The angulation and arching of the dorsal ribs suggest that these animals had a broad (barrel-like) torso. Intercostal uncinate plates were present, attached to the posterior margins of some of the largest dorsal ribs. Their attachment sites are clearly marked, and these plates might have been composed of calcified cartilage in larger individuals. The sacral vertebrae fuse progressively during ontogeny, in an anterior-to-posterior sequence. The sacral ribs are long and robust, and tilt the iliac blade outward dorsally. A sacricostal ‘yoke’ (created by the fusion of the distal ends of adjacent sacral ribs) never forms. The base of the tail has a unique ball-and-socket-style joint between the centra of caudal vertebrae 1 and 2 in only one skeleton. This might have permitted powerful, but controlled, movements of the tail as a defensive weapon (or increased flexibility at the base of the tail, which might have been necessary for reproduction). Caudal ribs are initially long, blade-shaped projections that gradually decrease in size and become stub-like remnants that persist as far back as the midtail (approximately caudal vertebra 25). Haemal arches (chevrons) disappear nearer to the distal end of the tail (approximately caudal vertebra 35). Ossified tendons are preserved as epaxial bundles that are clustered in the ‘axillary’ trough (between the neural spine and transverse processes on either side of the midline). Ossified tendons are restricted to the dorsal and sacral region. Flattened ossified tendons are fused to the sides of sacral neural spines. In life, the ossified tendons might have formed a low-angled trellis-like arrangement. Appendicular skeleton: The pectoral girdle comprises a long scapula, with a distally expanded blade. The proximal portion is expanded and supports an oblique promontory, forming an acromial process anteriorly and a thick, collar-like structure posteriorly above the glenoid. Between these two features is a shallow basin, bordered ventrally by a sutural edge for the coracoid. The scapula–coracoid suture remains unfused in large (5-m-long) individuals. The coracoid bears a discrete foramen and forms a subcircular dished plate, with the shallowest of embayments along its posterior edge. Clavicles are present as small fusiform bones attached to the acromial process of the scapulae and leading edge of each coracoid. A sternum was reported as ‘some partially ossified element of the endoskeleton’ Owen (1863: 13), but subsequent preparation of the skeleton has removed all trace of this material. The humerus is relatively long and has a prominent rectangular and proximally positioned deltopectoral crest. The ulna is robust and tapers distally, but there is no evidence of an olecranon process. The radius is more rod-like and terminates distally in an enlarged, subcircular and convex articular surface for the carpus. The carpus is represented by an array of five discoid carpals. The manus is pentadactyl and asymmetrical, with short, divergent metacarpals and digits that terminate in small, arched and pointed unguals on digits 1–3 (only). The phalangeal formula of the manus is 2-3-4-3-2. The pelvis is dominated by a long ilium; the preacetabular process is arched, transversely broad, and curves laterally. In juveniles, this process is short and horizontal, but during ontogeny it increases considerably in length and becomes arched. The iliac blade is tilted laterally, meaning that its dorsal blade partly overhangs the femur. The acetabulum forms a partial cupola, and there is a curtain-like medial wall that reduces the acetabular fenestra to a comparatively low, triangular opening between the pubis and ischium. The postacetabular portion of the ilium is long and supports a brevis shelf. The ischium has a long, laterally compressed shaft that hangs almost vertically beneath the ilium, and there is no obturator process. The pubis has a long, narrow shaft and a relatively short, deep, laterally compressed prepubic process that twists laterally (its distal end lies almost perpendicular to the long axis of the ilium). The articular pad on the pubis for the femoral head faces posteriorly. The obturator foramen is not fully enclosed within the pubis, but its foramen is closed off posteriorly by the pubic peduncle of the ischium. The femur is stout and has a slightly medially offset femoral head, and the greater trochanter forms a sloping shoulder continuous with, and lateral to, the femoral head. The anterior (lesser) trochanter is prominent and forms a thick, thumb-shaped projection on the anterolateral corner of the femoral shaft. The fourth trochanter is pendent and positioned at midshaft. In larger individuals, it appears to become thickened and reinforced by becoming coated with metaplastic bone derived from the tendons attached to its surface. The distal end of the femoral shaft is slightly curved and expands to form condyles. There is a deep and broad posterior intercondylar groove, but the anterior intercondylar groove is barely discernible in juveniles and not much better developed in subadults. The tibia and fibula are shorter than the femur. The tibia is structurally dominant, and the shorter fibula is comparatively slender and bowed. The proximal tarsals are firmly bound by connective tissue to the distal ends of the tibia and fibula. The distal end of the tibia is stepped, which aids the firm interlock between the crus and proximal tarsals. There appear to be two roughly discoid tarsals (distal tarsals 3 and 4), and a rudiment of distal tarsal 5 appears to be sutured to the lateral margin of distal tarsal 4. Five metatarsals are preserved, but the fifth is a splint of bone attached to the proximal end of metatarsal 4. Metatarsals 2–4 are dominant, long and are syndesmotically interlocked proximally, but their shafts splay apart distally. Metatarsal 1 is much shorter than the other three, but it retains two functional phalanges (including a short, pointed ungual). The foot is anatomically tetradactyl but functionally tridactyl. The pedal digit formula is 2-3-4-5-0. The digits diverge, but each appears to curve medially along its length, creating the impression of asymmetry. This asymmetry is emphasized, because the three principal unguals are also twisted medially. The ungual of digit 2 is the largest and most robust of the three, whereas that of digit 4 is the smallest and least robust. The general girth of the torso and the displacement of the abdomen posteriorly (a consequence of the opisthopubic pelvic construction in this dinosaur) constrained the excursion of the hindlimb during the protraction phase of the locomotor cycle. The anterolateral displacement of the hindlimb during protraction is in accord with the freedom of motion that is evident at the acetabulum, the susceptibility of the hindlimb to torsion between and within its component parts, and the asymmetry of the foot. It is probable that thyreophorans (notably, ankylosaurs) used a similar oblique-parasagittal hindlimb excursion to accommodate their equally large and wide abdomens. This surmise accords with the structure of the pelves and hindlimbs of ankylosaurs. Derived stegosaurs might have obviated this ‘problem’, in part, because their hindlimbs were longer and their torsos and abdomens narrower and capable of being ‘stretched’ vertically to a greater extent. Nevertheless, the structure of their acetabula and hindlimbs indicates that the oblique-parasagittal style of hindlimb excursion remained a possibility and might be an evolutionary remnant of the locomotor style of basal, shorter-limbed stegosaurs. A reconstruction of the endoskeleton of Scelidosaurus is presented on the basis of this updated description. Although quadrupedal, this animal was only facultatively so, judged by its forelimb-to-hindlimb proportions and structure; it therefore betrays bipedality in its ancestry.

Ammi majus (L.) is commonly used to cure many diseases in Moroccan folk medicine, especially vitiligo. This research tries to evaluate the phytochemical constituents of two aqueous extracts (E1; Maceration; 48 h) and (E2: Infusion; 1h) and three organic fractions (F1: Cyclohexane), (F2: Ethyl acetate (EtOAc)) and (F3: Ethanolic (EtOH)) of A. majus (L.) seeds, as well as to study the antioxidant and analgesic activity of the species. Phytochemical analysis, antioxidant activity (DPPH, FRAP, ABTS, and TAC tests), and analgesic activity (writhing and tail immersion were induced by Acetic acid tests) were analyzed according to the literature. A quantitative phytochemical study indicate that the E1 had the highest content of total polyphenols (26.95 ± 0.53 mg GAE/g extract) and flavonoids (37.92 ± 0.46 mg QE/g extract), while F3 showed a promising flavonol content (24.26±0.08 mg QE/g extract). Tannins were found to be high in F1 (59.27 ± 0.16 mg CE/g extract) and F2 (57.65 ± 1.18 mg CE/g extract). Antioxidant results reveals that DPPH (IC50 = 179.68 ± 0.47 μg/mL) and FRAP (EC50 = 367.03 ± 0.12 μg/mL) show to E1 a high antioxidant activity. Regarding the analgesic activity of the different studied extracts, it was found that E1 has a high peripheral analgesic effect with 62.32 % and a high central analgesic potential throughout the experimentation at 500 mg/kg. Our studies demonstrated for the first time that A. majus seeds extracts have high antioxidant and analgesic activities through different analysis techniques.

The study of language acquisition and instruction is not new to academics, but it never fails to excite linguists and teachers. The goal of Communicative Language Teaching (CLT) is to help students develop their communication skills so that they can effectively communicate in a target language. Since its start in the 1970s, when the need for language learners to improve their communication skills was rising, this approach has received worldwide reputation. But since many educators still reject this method, many worries remain. According to Chomsky (1957), the four main aspects of language acquisition lexis, syntax, phonology, and morphology are focused on linguistic competence. Hymes (1971) argues that pragmatic, sociolinguistic, semantic, and grammatical considerations are more important. When it comes to teaching second languages, the theories put forward by researchers, have been game-changers for communicative language instruction. The communicative language teaching (CLT) technique places an emphasis on students' active participation in second language classroom activities and provides more opportunities for students to improve their communication skills compared to the grammar-translation method. Other traditional methods of instruction do not typically use this component. In the context of ESL instruction, this article delves deeply into the ideas and methods of Communicative Language Teaching (CLT). In particular, it compares and contrasts conventional methods of instruction with Communicative Language Teaching (CLT) and gives a brief summary of its advantages and disadvantages. In addition, the article delves into the latest advancements in CLT and the difficulties encountered while applying CLT in an academic environment. After that, the post helps educators understand Communicative Language Teaching (CLT) better. Furthermore, it suggests possible outcomes, such as creating classroom activities and encouraging student motivation, of utilizing CLT to teach English in a university context. References Abahussain, M. O. (2016). Implementing Communicative Language Teaching Method in Saudi Arabia: Challenges Faced by Formative Year Teachers in State Schools. (PhD). University of Stirling, Scotland. Abrejo, B., Sartaj, S., & Memon, S. (2019). English Language Teaching through Communicative Approach: A Qualitative Study of Public Sector Colleges of Hyderabad, Sindh. Advances in Language and Literary Studies, 10(5), 43–49. https://doi.org/10.7575/aiac.alls.v.10n.5p.43 Ahn, S. Y., & Kang, H.-S. (2017). South Korean university students’ perceptions of different English varieties and their contribution to the learning of English as a foreign language. Journal of Multilingual and Multicultural Development, 38(8), 712-725. https://doi.org/10.1080/01434632.2016.1242595. Al-Nasser, A. S. (2015). Problems of English language acquisition in Saudi Arabia: An exploratory-cum-remedial study. Theory and Practice in Language Studies, 5(8), 1612- 1619. Alsalmi, A. A. (2014). Challenges confronting teachers of English language. (Master). Taif University, Taif. Bachman, L. (1990). Fundamental Considerations in Language Testing. Oxford: Oxford University Press. Batawi, G. H. (2007). Exploring the use of CLT in Saudi Arabia. (Master). American University of Sharjah, Sharjah. Borg, S. (2017). Teachers’ beliefs and classroom practices. In The Routledge handbook of language awareness, (pp. 93-109): Routledge. Brandl, K. (2019). Communicative Language Teaching in Action: Putting Principles to Work. Pearson Education, Inc. Brandl, K., & Bauer, G. (2002). Students’ Perceptions of Novice Teaching Assistants’ Use of the Target Language in Beginning Foreign Language Classes: Preliminary Investigation. In W. Davis, J. Smith, & R. Smith (Eds), Ready To Teach: Graduate Teaching Assistants Prepare for Today and for Tomorrow (pp.128-138). Stillwater, OK: New Forums Press Brown, H. D. (2014). Principles of Language Learning and Teaching (6th ed.). White Plains, NY Pearson Education. Canale, M. & Swain, M. (1980). Theoretical bases of communicative approaches to second language teaching and testing. Applied Linguistic, 1(1), 1-47. Chomsky, N. (1957). Syntactic structures. Mouton. Courtney, D. (2020). Activities to Activate and Maintain a Communicative Classroom. English Teaching Forum, 58(1), 10-21. Dey, M. (2021). Psychological processes in language learning and teaching: Scoping review and future research directions. Journal of Psychological Perspective, 3(2), 105-110. Dey, M. (2023). The primary characteristics of English pragmatics in Applied Linguistics: Exploring the Key Features of English Pragmatics in Applied Linguistics. Inverge Journal of Social Sciences, 2(2), 1–13. https://doi.org/10.1022/ijss.v2i2.25 Dey, M., Amelia, R., & Herawati, Y. W. (2023). Challenging the'Native Speaker'Ideal: The Impact of Native Speakerism on Language Education. Lingua Didaktika: Jurnal Bahasa dan Pembelajaran Bahasa, 17(2), 232-245. Dos Santos, L. M. (2016). Foreign language teachers' professional development through peer observation programme. English Language Teaching, 9(10), 39-46. https://doi.org/10.5539/elt.v9n10p39. Dos Santos, L. M. (2017). How do teachers make sense of peer observation professional development in an Urban School. International Education Studies, 10(1), 255-265. https://doi.org/10.5539/ies.v10n1p255. Dos Santos., L. M. (2019). Science lessons for non-science university undergraduate students: An application of visual-only video teaching strategy. Journal of Engineering and Applied Sciences, 14(1), 308–311. https://doi.org/10.36478/jeasci.2019.308.311. Dos Santos, L. M. (2020). The Discussion of Communicative Language Teaching Approach in Language Classrooms, Journal of Education and e-Learning Research, 2020. Journal of Education and E-Learning, 7(2), 104-109. Doughty, C., & Long, M. H. (2003). The handbook of second language acquisition. Blackwell Publishing. Harley, B., & Swain, M. (1984). The interlanguage of immersion and its implications for second language teaching. In A. Davies, C. Criper, & P. R. Howatt (Eds.), Interlanguage (pp. 291-311). Edinburgh: Edinburgh University Press. Huang, S.-H., & Yang, L.-C. (2018). Teachers’ Needs in the Advancement of Communicative Language Teaching (CLT) in Taiwan. TESOL International Journal, 13(1), 100-117. Holliday, A. (1994). Appropriate Methodology and Social Context. Cambridge: Cambridge University Press. Hymes, D. (1971). Pidginization and creolization of languages. London, UK: Cambridge University Press. Iwashita, N., & Ngoc, K. M. (2012). A comparison of learners’ and teachers’ attitudes toward communicative language teaching at two universities in Vietnam. University of Sydney Papers in TESOL, 7, 25-49. Jung, S. K., & Norton, B. (2002). Language planning in Korea: the new elementary English program. In Tollefson, J.W. (Ed.), Language policies in education: Critical issues (pp. 245-265). New Jersey: Lawrence Erlbaum Associates, Publishers. Kachru, B. (1992). World Englishes: approaches, issues and resources. Language Teaching, 25, 1–14. Kennedy, P. (2002). Learning cultures and learning styles: Myth-understandings about adult (Hong Kong) Chinese learners. International Journal of Lifelong Education, 21(5), 430-445. https://doi.org/10.1080/02601370210156745. Kiato, S. K., & Kiato, K. (1996). Testing Communicative Competence. The TESOL Internet Journal, 2(5). Larsen-Freeman, D. (2000). Techniques and principles in language teaching (2nd ed.). New York: Oxford University Press. Lee, J. S., & Lee, K. (2019). Perceptions of English as an international language by Korean English-major and non-English-major students. Journal of Multilingual and Multicultural Development, 40(1), 76-89. Li, D. (1998). It’s always more difficult than you plan and imagine: Teachers’ perceived difficulties in introducing the communicative approach in South Korea. TESOL Quarterly, 32(4), 677-703.Littlewood, W. (1981). Communicative language teaching: An introduction. Cambridge: Cambridge University Press. Lyster, R., & Ranta, L. (1997). Corrective feedback and learner uptake. Studies in Second Language Acquisition. https://doi.org/19. 10.1017/S0272263197001034. Natividad, M. R. A., & Batang, B. L. (2018). Students’ Perceptual Learning Styles and Attitudes toward Communicative Language Teaching. TESOL International Journal, 13(4), 104-120. Nunan, D. (1989). Designing tasks for the communicative classroom. Cambridge: Cambridge University Press. Nunan, D. (1991). Communicative tasks and the language curriculum. TESOL Quarterly, 25, 279–295. Ozsevik, Z. (2010). The use of communicative language teaching (CLT): Turkish EFL teachers’ perceived difficulties in implementing CLT in Turkey. (Unpublished doctoral dissertation). University of Illiois at Urbana-Champaign, USA. Pennycook, A. (1994). The cultural politics of English as an international language. London: Longman. Pham, H. H. (2007). Communicative language teaching: unity within diversity. ELT Journal, 61(3), 193-201. Phillipson, R. (1992). Linguistic imperialism. Oxford, England: Oxford University Press. Riggenbach, H., & Lazaraton, A. (1991). Promoting Oral Communication Skills. In M. Celce-Murcia (Ed.), Teaching English as a Second or Foreign Language (pp. 125-136). Los Angeles: University of California. Richards, J. C. (2006), Communicative language teaching today. New York: Cambridge University Press. Richards, J. C., & Rodgers, T. (2001). Approaches and methods in language teaching. Cambridge: Cambridge University Press. Savignon, S. J. (1997). Communicative competence: Theory and classroom practice (2nd ed.). Sydney, NSW: McGraw-Hill. Saengboon, S. (2002). Beliefs of Thai EFL teachers about communicative language teaching. (Doctoral dissertation). Indiana University Bloomington, USA. Schulz, R.A. (1996). Focus on form in the foreign language classroom: Learners’ and teachers’ view on error correction and the role of grammar. Foreign Language Annals, 29(3), 333-364. Sawalmeh, M. H., & Dey, M. (2023). Globalization and the increasing demand for spoken English teachers. Research Journal in Advanced Humanities, 4(2). Spada, N., & Lightbown, P. M. (1989). Intensive ESL programs in Quebec primary schools. TESL Canada Journal, 7, 11-32. Souriyavongsa, T., Rany, S., Abidin, M. J. Z., & Mei, L. L. (2013). Factors causes students low English language learning: A case study in the National University of Laos. International Journal of English Language Education, 1(1), 179-192. Thompson, G. (1996). Some misconceptions about communicative language teaching. ELT Journal, 50(1), 9–15. Tomlinson, B. (2001). Humanising the Coursebook. Humanising Language Teaching, 3(5). Walia, D. N. (2012). Traditional teaching methods vs. CLT: A study. Frontiers of language and teaching, 3(1), 125-131. Weiner, L. (2012). The future of our schools: Teachers unions and social justice. Chicago, IL: Haymarket Books. Weiner, L., & Jerome, D. (2016). Urban teaching: The essentials (3rd ed.). New York, NY: Teachers College Press. West, A. J. (2016). Adaptation of Communicative Language Teaching Methodology to an English Textbook for English Language Learning of NIDA Students. PASAA, 52, 25-52 Widdowson, H. G. (1990). Aspects of Language Teaching. Oxford: Oxford University Press. Zhang, J. L. (2006, November 11-13). The ecology of communicative language teaching: Reflecting on the Singapore experience [Paper presentation]. Annual CELEA International Conference: Innovating English Teaching: Communicative Language Teaching (CLT) and Other Approaches, China English Language Education Association (CELEA) and Guangdong University of Foreign Studies, Guangzhou, China.

AbstractA plasma immersion ion implantation (PIII) hydrogenation process using an inductively-coupled plasma (ICP) source is implemented for defect passivation in polycrystalline silicon (poly-Si) thin film transistors (TFT's). Device parameter improvement saturates in 4 minutes, which is considerably shorter than for other reported hydrogenation methods. Stress test indicates that the devices hydrogenated by this novel technique have much better long-term reliability. The hydrogenation effects on two types of trap states are analyzed the current-voltage characteristics of the devices. The densities of deep states and tail states are significantly reduced after short time hydrogenation.

AbstractA plasma immersion ion implantation (Pill) hydrogenation process using an inductively-coupled plasma (ICP) source is implemented for defect passivation in polycrystalline silicon (poly-Si) thin film transistors (TFT's). Device parameter improvement saturates in 4 minutes, which is considerably shorter than for other reported hydrogenation methods. Stress test indicates that the devices hydrogenated by this novel technique have much better long-term reliability. The hydrogenation effects on two types of trap states are analyzed the current-voltage characteristics of the devices. The densities of deep states and tail states are significantly reduced after short time hydrogenation.

Padmak Agad is indicated in all types of Luta and Keet Visha. According to Acharya Vagbhatta, heaviness in head and eyes, coma, dizziness, dyspnoea, pain, swelling, fever, itching and anorexia are common symptoms of all Keeta Damsha. Several synthetic and plant origin analgesic are being tested for their efficacy and potency on different animal model including hot plate, tail flick, tail clip, cold pain, filament pain, tail immersion technique, acetic acid induced writhing test, formalin induced writhing test etc. Material and Methods: Tail clip model and Hot plate model were selected for the present study. Eighteen Swiss albino mice had been divided in to three groups each group contain six mice. First group was negative control group; second group was test drug group while third group was standard group. Test drug was given to the group 2 for 7 days O.D. Result: When we performed the tail clip test, response time of group 1, 2, 3 were 4.40±1.853, 14.07±9.309, 40.68±11.759 seconds respectively. In Hot Plate model, calculated response time of group 1, 2, 3 were 2.93±0.667, 5.46±0.911, 6.04±0.857 seconds respectively. When we used Dunnett's multiple comparisons test to compare the group 1 to group 2 and group 1 to group 3, there was statistically no significant role of test drug in tail clip model and hot plate model. Discussion and Conclusion: No effect of Padmak Agad is found in tail clip model. Padmak Agad is effective in hot plate model but not up to significant level.

AbstractHollow organs such as the lungs pose a considerable challenge for post-mortem imaging in preclinical research owing to their extremely low contrast and high structural complexity. The aim of our study was to enhance the contrast of tuberculosis lesions for their stratification by 3D x-ray–based virtual slicing. Organ samples were taken from five control and five tuberculosis-infected mice. Micro-Computed Tomography (CT) scans of the subjects were acquired in vivo (without contrast agent) and post-mortem (with contrast agent). The proposed contrast-enhancing technique consists of x-ray contrast agent uptake (silver nitrate and iodine) by immersion. To create the histology ground-truth, the CT scan of the paraffin block guided the sectioning towards specific planes of interest. The digitalized histological slides reveal the presence, extent, and appearance of the contrast agents in lung structures and organized aggregates of immune cells. These findings correlate with the contrast-enhanced micro-CT slice. The abnormal densities in the lungs due to tuberculosis disease are concentrated in the right tail of the lung intensity histograms. The increase in the width of the right tail (~376%) indicates a contrast enhancement of the details of the abnormal densities. Postmortem contrast agents enhance the x-ray attenuation in tuberculosis lesions to allow 3D visualization by polychromatic x-ray CT, providing an advantageous tool for virtual slicing of whole lungs. The proposed contrast-enhancing technique combined with computational methods and the diverse micro-CT modalities will open the doors to the stratification of lesion types associated with infectious diseases.

Dracaena reflexa (family: Asparagaceae), commonly known as Song of India is a tropical plant that is traditionally claimed to have high medicinal values. The current exploration was intended to assess the pain-relieving and anti-inflammatory potentials of methanolic extract of Dracaena reflexa (MEDR) leaves in rodents. Acute oral toxicity studies were performed for the MEDR as per OECD guidelines 423. Anti-inflammatory action was assessed by the carrageenan incited paw edema method. The anti-nociceptive effect was explored by using acetic acid incited writhing responses, tail immersion, and hot plate technique in rodents. Acute oral toxicity investigation for 14 days reveals the safety of MEDR with no mortality at 2000 mg/kg. Hence 200 & 400 mg/kg was selected for in vivo studies. MEDR at both doses exhibited significant anti-inflammatory action by diminishing paw volume (ml) (0.29±0.016, 0.17±0.014) when compared to control (1.71±0.015) at the 4th hour of the study. Acetic acid incited writhing technique indicates the anti-nociceptive effect as the number of writhing responses with leaf extract significantly decreased (12.33±0.57) in comparison to control rats (27.33±1.52).

Abstract Background A. capitiformis (Poir.) Ooststr has a long history of usage as a medicinal cure for a wide variety of illnesses in many different cultures. Pharmacological properties and phytochemical characterization of the crude A. capitiformis whole plant are evalutted, in this paper. Methods Antioxidant activity was tested by the DPPH free radical scavenging method. In vitro anti-arthritic, anti-inflammatory, and cytotoxic effects were assessed using Bovine serum albumin (BSA), protein denaturation method, and brine shrimp mortality assays, respectively with antihelmintic activity through Pheretima Posthuma worms. Acetic acid-induced writhing, hot plate and tail immersion testing assessed in vivo analgesia. CNS activity was evaluated through elevated plaze maize, open field, hole cross, and head dipping method. Results Phytochemiical investigation of A. capitiformis showed the presence of alkaloid, saponin, terpennoids, steroid and flavonoids etc. with the % yield of crude 2.04%.With an IC50 of 45.35 µg/ml, the whole plant methanolic preparation has antioxidant activity equivalent to ascorbic acid. Anti-arthritic protein blocking dropped from 74.25 ± 0.12% to 12.18 ± 0.12%. 1000 µg/ml extract demonstrated 54.05 ± 0.12*% anti-inflammatory activity with protein denaturation. In the cytotoxicity assay, the extract had 129.72 µg/ml LC50 and the positive group 34.67 µg/ml. Unlike Albendazole, the methanol extract triggered mature earthworms at 50 mg/ml. The extract’s analgesic efficacy at 200 and 400 mg/kg was statistically significant (p < 0.001) in the acetic acid writhing and tail immersion method. The hot plate technique yielded statistically significant results only at 400 mg/kg (p < 0.001). Only 400 mg/kg was statistically significant in the Elevated Plaze Maize and Hole Board Procedure (p < 0.01). The hole cross and open field methods yielded highly statistically significant outcomes at 200 and 400 mg/kg (p < 0.001). Conclusion In this research, the whole crude methanol extract of A. capitiformis revealed phytochemicals, antioxidants, in vitro anti-inflammatory and anti-arthritic properties, cytotoxicity, anti-helminthic, in vivo analgesic, and CNS inhibitory activities.

Objectives: The current study aimed to conduct a phytochemical screening of commonly known fruit red grape ( Vitis vinifera L.) seed methanolic extract through gas chromatography and mass spectrometry (GC-MS) to identify the bioactive compounds responsible for its health benefits and evaluate the pharmacological potentialities of the extract and its fractions against oxidation, inflammation, pain, and diarrhea. Methods: The in vitro antioxidant, anti-inflammatory, and cytotoxic characteristics of methanolic extracts and various solvent fractions of V. vinifera were evaluated using the DPPH free radical scavenging assay, membrane stabilizing, and brine shrimp lethality bioassay. Furthermore, the study assessed the effects of crude extracts (200, 400, and 600 mg/kg of body weight) on pain relief and reduction of diarrhea in animals using methods such as tail immersion, the acetic acid-induced writhing technique, and a diarrheal mouse model induced with castor oil. Results: A total of 73 phytoconstituents were predominantly found in the seed extract based on the GC-MS analysis. Among the identified compounds, 9-octadecenamide (13.7%), and ( 9E,11E)-octadeca-9,11-dienoate (11.07%) are most abundant. Several notable constituents, such as gamma-sitosterol, stigmasterol, paromomycin, 4,6-cholestadienol, gamma-tocotrienol, 24-Propylidenecholest-5-en-3beta-ol, and alpha-tocopherol acetate, are also present. The methanolic extract of V. vinifera seed and its different solvent fractions showed promising antioxidant properties (IC50 = 1.19-17.42 µg/mL) compared to the standard antioxidant butylated hydroxytoluene (IC50 = 20.46 µg/mL). Aqueous soluble fraction exerted inhibition of nearly 50% heat-induced hemolysis compared to the standard acetylsalicylic acid (42%). Besides, all the tested doses (200, 400, and 600 mg/kg bw) of the crude extract showed significant ( P < .05) analgesic and antidiarrheal effects. Conclusion: The current findings endorsed the health benefits of V. vinifera by revealing potent antioxidant, anti-inflammatory, analgesic, and antidiarrheal effects. Nevertheless, further in-depth analysis of the plant’s chemical constituents and pharmacological effects on health is warranted for novel drug discovery from V. vinifera.

Introduction: Natural plant-based medicines have gained popularity recently as a major source of inventive, risk-free, and more potent secondary bioactive compounds with medicinal potential. Catharanthus ovalis is a perennial shrub containing various indole alkaloids cultivated extensively for local medical purposes.Methods: This research is conducted to identify the phytocompounds present in the leaves of C. ovalis and its central and peripheral analgesic, thrombolytic, and membrane-stabilizing activities through tail immersion, acetic acid-induced writhing, human blood clot lysis, and erythrocyte lysis by heat and hypotonic solution methods, respectively.Results and discussion: A total of 39 compounds were identified using GC–MS/MS techniques, including hexadecanoic acid, methyl ester (56.749%), methyl stearate (29.782%), carvacrol and its TBDMS derivative (12.586%), and 9-octadecenoic acid, methyl ester, (E)-] (9.297%) presented in high quantity. The highest tail immersion latency was observed for the 600 mg/kg extract of C. ovalis crude extract. Both 400 and 600 mg/kg doses of C. ovalis crude extract exhibited prominent peripheral analgesic activity. The maximum thrombolytic effect was observed by DCM soluble fraction extract by inhibiting 54.87% of the clot. However, the aqueous-soluble fraction of this extract manifested an excellent membrane-stabilizing effect by showing 73.98% and 87.51% hemolysis against heat- and hypotonic-induced hemolysis, respectively. Some of the compounds were identified as active agents against different receptors related to these diseases, which supported the findings of in vitro and in vivo tests.Conclusion: Further investigation needs to be conducted to specify and identify the exact mechanism of action of these compounds.

Background: Pain is a disturbing sensory and emotive sentiment triggered mainly by tissue-damaging stimuli. This study aimed to evaluate the potential effect of tramadol and nefopam on acute pain. Methods: Thirty Sprague-Dawley rats (each 200-250 g) were randomly allocated into three sets (n=10). The nefopam group was treated with nefopam (3.5 mg/kg) by intraperitoneal (IP) injection; the tramadol group was given tramadol (50mg/kg) by IP injection, and the control group was treated with normal saline. Two main methods were proposed for assessing and monitoring rat pain: hot plate and tail-flick techniques using tail immersion. Results: It was revealed a significant change (p<0.0001) in the outcome in the animal groups that received nefopam (3.5mg/kg/IP) and tramadol (50mg/kg/IP) in the prospect of hot plate test (hand paw lick parameter) and tail flick test. Regarding the hot plate test (jumping parameter), there was no significant change (p>0.05) in animals treated with tramadol and nefopam compared to the control group. Moreover, a considerable difference between the hot plate test (hand paw lick parameter) and the tail-flick test was detected between tramadol and nefopam-treated groups. However, no significant variance (P=0.101) was detected between the two groups in the hot plate test (jumping parameter). Conclusion: Tramadol showed better analgesic activity over nefopam in suppressing pain stimuli in acute settings with modest to severe pain, making tramadol a favourable choice for short-term management of postoperative pain.

AbstractElectrochromic smart windows (ESWs) can significantly reduce energy consumption in buildings, but their cost‐effective, large‐scale production remains a challenge. In this study, the instability of black phosphorus is leveraged to induce the growth of the tungsten oxide film through its decomposition process, inspired by the 2D material‐assisted in situ growth (TAIG) method. This approach results in the preparation of large‐scale, high‐performance WO3‐x·nH2O (n < 2) films. Characterization techniques and DFT calculations confirm efficient regulation of structural water and oxygen vacancies during TAIG preparation. The WO3‐x·nH2O films exhibit excellent electrochromic (EC) properties, including high transmittance modulation (74.2%@1100 nm), fast switching time (tc = 5.5 s, tb = 3.8 s), high coloration efficiency (124.7 cm2 C−1), and superior cyclic stability (transmittance modulation retained 94.7% after 20 000 cycles). Ultra‐large WO3‐x·nH2O film are prepared via a simple immersion process, and fabricated into a large‐area ESW under facile laboratory conditions, demonstrating the economic and practical feasibility of this approach in industrial‐scale production. Operated by the intelligent control circuit, the ESW exhibits remarkable EC properties and cyclic stability This research represents a milestone in improving the performance and industrial‐scale production of ESWs, bridging the gap to the commercialization of EC technology.

AbstractThe involvement of pro-inflammatory mediators complicates the complex mechanism in neuropathic pain (NP). This study investigated the roles of bromelain against pro-inflammatory mediators as a mechanism that underpins its antinociceptive and anti-anxiety effects in the peripheral model of NP. Sixty-four male Wistar rats randomly divided into eight groups, were used for the study. A chronic constriction injury model of peripheral neuropathy was used to induce NP. Tail-immersion and von Frey filaments tests were used to assess hyperalgesia while open field and elevated plus mazes were used to assess anxiety-like behaviour. NF-кB, iNOS, nitrate, and pro-inflammatory cytokines were investigated in the plasma, sciatic nerve, and brain tissues using ELISA, spectrophotometer, and immunohistochemistry techniques after twenty-one days of treatment. Bromelain significantly (p < 0.05) improved the cardinal signs of NP and inhibited anxiety-like behaviours in ligated Wistar rats. It mitigated the increases in cerebral cortex interleukin (IL) -1β, IL-6, and PGE2 levels. Bromelain reduced NF-кB, IL-1β, IL-6, TNF-α, PGE2, and nitrate concentrations as well as the expression of iNOS in the sciatic nerve. Hence, the antinociceptive and anxiolytic effects of bromelain in the sciatic nerve ligation model of NP is in part due to its ability to reduce nitrosative and inflammatory activities.

ABSTRACTIn this study, we explored the eco‐friendly synthesis of photoluminescent CCDs employing a direct one‐step pyrolysis process, utilizing natural Cortex Phellodendri Chinensis as the precursor material and studied their analgesic effect in mice. The synthesized carbon dots underwent comprehensive characterization through a range of spectroscopic and microscopic techniques. These included UV‐Vis, FTIR, fluorescence spectroscopy and HR‐TEM, DLS instruments. HR‐TEM results exhibited the presence of homogenous spherical‐shaped C‐dots of about 3.3 nm without aggregates. Furthermore, the prepared CCDs were studied for their in vivo analgesic effect in mice by performing tail‐immersion, hot plate and acetic acid writhing tests. Also, an MTT assay was performed to assess the in vitro cytotoxicity of CCDs against L929 cells. In vitro cytotoxicity studies revealed that L929 cells exhibited higher cell viability when treated with prepared CCDs. The cellular uptake studies revealed the phase contrast images of MG‐63 cells at wavelength 488 nm clearly depicted the aggregation of green, fluorescent CCDs within the cells while leaving nuclei unobscured. In addition, to the best of our understanding, the results presented in this paper showed that CCDs exhibited an important analgesic effect and enhanced anti‐nociceptive activity, which may be due to stimulation of the opioidergic system. Consequently, CCDs appear to be a viable analgesic alternative for traditional analgesic candidates in pain management.

In this study, the anti-nociceptive potential of Schiff bases derivatives of 4-aminoantioyrine, (Z)-4-(4-hydroxy-3-methoxybenzylideneamino)-2, 3-dimethyl-1-phenyl-1, 2dihydropyrazol-5-one 1 and (Z)-4-(2-nitrobenzylideneamino)-2, 3-dimethyl-1-phenyl-1-2-dihydropyrazol-5-one 2 were tested in various mice pain models and their binding affinities with different drug targets were evaluated through molecular docking studies. The binding scores were calculated through molecular docking techniques for receptor sensitivity. Acute toxicity test suggests the safety of both compounds up 200 mg/kg. In the righting test, compound 1 and 2 had a significant effect in a dose-dependent manner and showed 59.46% and 48.40% blockade of pain at 150 mg/kg, respectively. In the formalin test, dose-dependently compound 1 showed 52.95% and 62.02% of inhibition in the early and late phase at 150 mg/kg. Similarly, Compound 2 showed 45.74% and 55.95% inhibition in the early and late phases at 150 mg/kg, respectively. In the tail immersion test, both compounds caused significant pain inhibition during various assessment times with maximum effects at 74.94% and 66.80% for 1 and 2 respectively at 150 mg/kg after 120 min. In molecular docking studies, compounds 1 and 2 showed a greater affinity for LOX with a docking score of –6.50 and 6.57 respectively. Similarly, for compounds 1 and 2 the docking was –4.94 and –4.83 with COX-1 while –5.10 and –4.85 with COX-2, respectively. Taken together, both the compounds exhibited marked antinociceptive effects in various pain-induced models possibly mediated by inhibition of LOX and COX pathways.

Typha domingensis, a medicinal plant with significant traditional importance for curing various human diseases, has potentially bioactive compounds but was less explored previously. Therefore, this study aims to investigate the therapeutic potential of T. domingensis by evaluating the phytochemical profile through high-performance liquid chromatography (HPLC) techniques and its biological activities (in vitro and in vivo) from the methanolic extract derived from the entire plant (TDME). The secondary metabolite profile of TDME regulated by reverse phase ultra-high-performance liquid chromatography–mass spectrometry (RP-UHPLC–MS) revealed some bioactive compounds by -ve and +ve modes of ionization. The HPLC quantification study showed the precise quantity of polyphenols (p-coumaric acid, 207.47; gallic acid, 96.25; and kaempferol, 95.78 μg/g extract). The enzyme inhibition assays revealed the IC50 of TDME as 44.75 ± 0.51, 52.71 ± 0.01, and 67.19 ± 0.68 µgmL-1, which were significant compared to their respective standards (indomethacin, 18.03 ± 0.12; quercetin, 4.11 ± 0.01; and thiourea, 8.97 ± 0.11) for lipoxygenase, α-glucosidase, and urease, respectively. Safety was assessed by in vitro hemolysis (4.25% ± 0.16% compared to triton × 100, 93.51% ± 0.36%), which was further confirmed (up to 10 g/kg) by an in vivo model of rats. TDME demonstrated significant (p < 0.05) potential in analgesic activity by hot plate and tail immersion tests and anti-inflammatory activity by the carrageenan-induced hind paw edema model. Pain latency decreased significantly, and the anti-inflammatory effect increased in a dose-dependent way. Additionally, in silico molecular docking revealed that 1,3,4,5-tetracaffeoylquinic acid and formononetin 7-O-glucoside-6″-O-malonate possibly contribute to enzyme inhibitory activities due to their higher binding affinities compared to standard inhibitors. An in silico absorption, distribution, metabolism, excretion, and toxicological study also predicted the pharmacokinetics and safety of the chosen compounds identified from TDME. To sum up, it was shown that TDME contains bioactive chemicals and has strong biological activities. The current investigations on T. domingensis could be extended to explore its potential applications in nutraceutical industries and encourage the isolation of novel molecules with anti-inflammatory and analgesic effects.

Authors: Meta Rath, Roland Nitschke, Alida Filippi, Olaf Ronneberger & Wolfgang Driever ### Abstract In this protocol we describe a method to produce multi-view confocal 3D datasets suitable to be processed by the Virtual Brain Explorer (ViBE-Z) software. The method is optimized for Zebrafish (Danio rerio) embryos and larvae from one to five days post fertilization, but may be used also for other small biological objects. Zebrafish larvae are stained using either fluorescent in situ hybridization or immunostaining. In addition, all samples are counterstained with a nuclear stain to generate information to be used for anatomical reference. Stained larval brains are imaged using standard laser scanning confocal microscopes. To properly represent regions of very high as well as very low signal intensity we generate image stacks at different laser intensities and merge them to high dynamic range datasets. Further, multiple views are recorded and merged into high resolution combined datasets. To reduce the loss of information by restricted optical depth as a result of absorption and light scattering occurring in thick samples, image stacks are recorded both from the dorsal and ventral side of larvae. Both dorsal and ventral recordings are fused using attenuation correction of the ViBE-Z software, leading to a data representation that significantly reduces absorption and diffraction artifacts typical for microscopy of tissues deep inside biological samples. ### Introduction Confocal imaging of thick biological objects like the larval zebrafish brain, which is 400 to 500 µm in each dimension during larval stages, suffers from absorption and light scattering. The loss of signal and increased noise make high resolution imaging at deep optical planes difficult. Several optical techniques aim at reducing these problems, including multi-view optical section microscopy (Huisken et al., 2004), two-photon microscopy (Helmchen and Denk, 2005), and stitching of individual stacks to a larger dataset (Emmenlauer et al., 2009). We recently developed an imaging framework to improve recording and analysis of confocal datasets of the larval zebrafish brain, the Virtual Brain Explorer for zebrafish (ViBE-Z; Ronneberger et al., 2012). ViBE-Z software also enables expression colocalization as well as anatomical analysis in zebrafish larval brains at single cell resolution. ViBE-Z requires high quality and high resolution confocal datasets. For this purpose confocal laser scanning microscopy is perfectly suitable as it is widely used, highly standardized and generates high spatial resolution images. We provide in this protocol a method to obtain high quality image stacks from zebrafish larval brain, using standard commercial confocal microscopes. To optimize the quality of the 3D volume data, we combine information from several optimized confocal image stacks. For best documentation of the broad range of signal intensities, high dynamic range imaging was performed by recording the samples at two different excitation laser light intensities. In order to obtain the best resolution deep inside the brain, larvae were imaged from two sides, dorsal and ventral, turning the mounted embryo on the stage. ViBE-Z enables the stitching of the two image stacks and use of the combined information for attenuation correction (Ronneberger et al., 2012). Using a 25x multi-immersion objective with a numerical apperture of 0.8 allowed to record a large part of the zebrafish brain in one stack. However, the method also enables imaging of the whole brain, by making one set of dorsal and ventral recording of the fore- and midbrain, and an additional set of the hindbrain. ViBE-Z fuses all image stacks to one high resolution data volume. The specific signal is generated by detecting antigens by fluorescent immunostaining (Holzschuh et al. 2003) or specific RNAs by fluorescent in situ hybridization (Filippi et al. 2007). In addition to the specific stain of the gene or antigen of interest, all cell nuclei of the larva were counterstained using nucleic acid dyes – either TOTO®-3 or Sytox® – to obtain a morphological and anatomical reference. In the following, we describe a step-by-step protocol, starting with the generation of larval samples followed by staining procedures, embedding of the larvae, and finally a description of the detailed confocal recording procedure. ### Reagents **Chemicals** 1. agarose (Bioron, Cat. No. 604005) - Blocking Reagent (Roche Applied Science, Cat. No. 11096176001) - BSA (bovine serum albumin) proteinase free (Sigma-Aldrich, Cat. No. A3059) - cyanoacrylate glue (“crazy glue” or “Sekundenkleber”) - DIG RNA labeling mix (Roche Applied Science, Cat. No. 11277073910) - dimethyl sulfoxide – DMSO (AppliChem, Cat. No. A3006) - disodium hydrogen phosphate – Na2HPO 4 (AppliChem, Cat. No. A1046) - formamide (AppliChem, Cat. No. A2156) - glycerol – C3H8O3 (AppliChem, Cat. No. A1123) - goat serum (PAA Laboratories, Cat. No. B11-035) - HEPES (Carl Roth GmbH, Cat. No. 9105) - methanol – CH4O (AppliChem, Cat. No. A3493) - methylene blue (Sigma-Aldrich, Cat. No. MB1) - mparaformaldehyde – PFA (Sigma-Aldrich, Fluka, Cat. No. 76240) - mphenylthiourea – PTU (Sigma-Aldrich, Cat. No. P-7629) - mpotassium chloride – KCl (Carl Roth GmbH, Cat. No. 6781) - mpotassium dihydrogen phosphate – KH2PO4 (AppliChem, Cat. No. A3620) - proteinase K (AppliChem, Cat. No. A3830) - sodium chloride – NaCl (AppliChem, Cat. No. A3597) - sodium hydroxide – NaOH (AppliChem, Cat. No. A3910) - sodium dihydrogen phosphate – NaH2PO4 (Carl Roth GmbH, Cat. No. K300) - Sytox Green (Invitrogen, Cat. No. S7020) - TOTO-3 iodide (Invitrogen, Cat. No. T3604) - Tris – C4H11NO3 (AppliChem, Cat. No. A2264) - tri sodium citrate – Na3C6H5O7 (Carl Roth GmbH, Cat. No. 3580) - TSA - Tyramide Signal Amplification Kit (Invitrogen, Cat. No. T20922) - Tween20 – polyoxyethylen 20 sorbitan monolaurate (AppliChem, Cat. No. A1389) - modeling clay (plastic) **Antibodies** Primary antibodies: 1. anti-3A10 (mouse; 1:50; Developmental Studies Hybridoma Bank) - anti-acetylated tubulin (mouse; 1:1000; Sigma-Aldrich, Cat. No. T7451) anti-digoxigenin peroxidase conjugated (sheep; 1:400; Roche Applied Science, Cat. No. 11093274910) - anti-GFP (chicken; 1:400; Invitrogen, Cat. No. A10262) Secondary antibodies: 1. anti-chicken Alexa 488 (goat; 1:1000; Invitrogen, Cat. No. A11039) - anti-mouse Alexa 488 (goat; 1:1000; Invitrogen, Cat. No. A11001) - anti-rabbit Alexa 633 (goat; 1:1000; Invitrogen, Cat. No. A21070) **Preparation of solutions** 1. Egg-water: 0.3 g/l sea salt in Millipore Milli-Q water - Methylene blue egg-water: 0.5-2ppm methylene blue in egg-water - 20x PBS stock solution: 35 mM KH2PO4, 208 mM NaH2PO4, 54 mM KCl, 2.74 M NaCl, dissolved in 1L Millipore Milli-Q water - 1x PBS working solution: 50ml 20xPBS stock solution in 1 L Millipore Milli-Q water, adjust pH with NaOH to 7.5 - 1x PBST working solution: 50 ml 20xPBS stock solution in 1 L Millipore Milli-Q water, 0.1% Tween 20, adjust pH with NaOH to 7.5 - 10x PTU stock solution: 2 mM PTU in reverse osmosis water, dissolved over night at 4°C, stored at 4°C - 1x PTU working solution in Methylene blue egg-water: 100 ml 10x PTU stock solution in Methylene blue egg-water - PFA 4%: 20 g PFA in 500 ml 1x PBS - proteinase K stock solution 20 mg/ml in Millipore Milli-Q water - proteinase K working solution: 10 µg/ml in PBST - 20x SSC stock solution: 3 M NaCl, 300M NaCitrate in 1 L Millipore Milli-Q water, adjust pH with HCl to 7.0 - 2x SSCT: 100 ml 20x SSC stock solution, 0.1% Tween 20 in 1 L Millipore Milli-Q water - 0.2x SSCT: 10 ml 20x SSC stock solution, 0.1% Tween 20 in 1 L Millipore Milli-Q water - Hybridization mix: 50% formamid, 5x SSC, 5 mg/ml Torula RNA, 50 µg/ml Heparin, 0.1% Tween 20 in 1 L Millipore Milli-Q water - TNT: 1 M TrisHCl pH 7.5, 1 M NaCl, 0.5% Tween 20 in 0.5 L Millipore Milli-Q water - TNTB: 1% blocking reagent in TNT - PBTD: 1% in DSMO in PBST - Blocking solution: 5% goat serum, 1% blocking reagent, 1% BSA proteinase free in PBTD - TOTO-3 iodide working solution: 1:2000 diluted in PBST - Sytox Green working solution: 1:30000 diluted in PBST - Mounting medium: 80% glycerol, 1% agarose in PBS - Goat serum: heat inactivated before use for 2 h at 56°C in a water bath ### Equipment 1. hollow needle 0.6×30 mm, size 14 (Braun, Melsungen, Germany) - cover slip 24×60 mm (Menzel-Glaeser, Braunschweig, Germany) - cover slip 22×22 mm (Menzel-Glaeser, Braunschweig, Germany) - cover slip 18×18 mm (Menzel-Glaeser, Braunschweig, Germany) - aluminium mounting frame holding 24×60 mm cover slip in 26×76 mm aluminium - frame (standard glass slide size; custom made) - Incubators (Heraeus Heracool 40, Kendro Laboratory Products, Asheville, NC, USA) - Turning Wheel (test-tube-rotator 34528, Snijders scientific b.v., Tilburg, Holland) - Zeiss LSM510-i-NLO (Carl Zeiss MicroImaging GmbH, Jena, Germany) (or other confocal microscope) - Objective: LD LCI Plan-Apo 25x / N.A. 0.8 Imm Korr multi immersion objective (or similar high aperture multi-immersion objective) ### Procedure **I. Embryo incubation and fixation** Zebrafish breeding and maintenance were carried out under standard conditions (Westerfield, 2000), larvae were raised in petri dishes in methylene blue egg-water. All subsequent incubations may be performed in 1.5 ml micro-centrifuge tubes. 1. To prevent pigmentation, incubate living embryos older than one day in egg-water containing 0.2 mM PTU until they reach the desired stage. - Fix larvae at the desired stage in 4% PFA in PBS overnight at 4°C. - Wash larvae 5 times 5 min in PBST. - Dehydrate larvae stepwise with increasing concentrations of methanol (5 min washing steps each of 25%, 50%, 75% MeOH in PBST and 100% MeOH). - Dehydrated larvae can be stored in MeOH at -20°C until they are used for staining. **II. Staining procedures** Larvae can be stained by immunohistochemistry (A) or in situ hybridization (B) and counterstained with a nuclear stain. All incubations may be performed in 1.5 ml microcentrifuge tubes. A. Fluorescent immunohistochemistry (IHC) IHC was carried out as reported (Holzschuh et al., 2003). 1. Rehydrate larvae with stepwise decreasing concentrations of methanol (5 min washing steps each 75%, 50%, and 25% MeOH in PBST). - Wash larvae 3 times 5 min in PBST. - (non-obligatory step: for embryos and larvae older than two days, some immunostains are improved by limited proteinase K digestion. Please determine optimal incubation times for each antigen. Suggested: digest larvae for 30 min (48 hpf larvae), 45 min (72 hpf larvae) or 60 min (96 hpf larvae) with proteinase K solution. Wash once with PBST; fix larvae again 20 min with 4% PFA (“post-fix”). Wash larvae 5 times 5 min with PBST.) - Block larvae one hour in blocking solution. - Incubate with primary antibody diluted in blocking solution overnight at 4°C. - The following day, wash larvae several times for 30 min in PBTD. - Incubate larvae overnight with the appropriate secondary antibody (diluted 1:1000 in PBTD + 1% Blocking Reagent; incubate in the dark). - On the third day, wash larvae 4 times 15 min in PBTD. - Wash larvae 4 times 15 min in PBST. B. Fluorescent in situ hybridization (FISH) FISH was performed as described in (Filippi, 2007). 1. Rehydrate larvae with stepwise decreasing concentrations of methanol (5 min washing steps each 75%, 50%, and 25% MeOH in PBST). - Wash larvae 3 times 5 min in PBST. - Bleach larvae 20 min with 1% H2O2 in PBST. - Wash larvae 2×5 min with PBST. - Digest larvae for 30 min (48 hpf larvae), 45 min (72 hpf larvae) or 60 min (96 hpf larvae) with proteinase K solution. - Wash once with PBST. - Fix larvae again 20 min with 4% PFA (“post-fix”). - Wash larvae 5 times 5 min with PBST. - Pre-hybridize larvae for at least 2 hours in hybridization mix at 65°C. - Hybridize larvae overnight in hybridization mix containing the specific digoxigenin-labeled RNA antisense probe at 65°C. - The following day the, wash larvae several times at 65°C: 1×20 min in hybridization mix; 2×20 min in 50% formamide in 2x SSCT; 1×20 min in 25% formamide in 2x SSCT; 2×20 min in 2x SSCT; 3×30 min in 0.2x SSCT - Wash larvae 5 min in TNT buffer at room temperature. - Block larvae in TNTB for at least 1 hour. - Incubate larvae overnight with a peroxidase-conjugated anti-digoxigenin antibody at 4°C. - On the third day, wash 5×15 min with TNT. - Stain larvae according to the TSA kit instructions (Invitrogen). The staining was carried out in the dark for 1 hour. - Wash larvae 3×5 min in TNT. **III. Nuclear staining** In order to visualize the morphological structures of the larvae, cell nuclei were stained either with TOTO-3 iodide or with SYTOX Green. 1. Incubate stained (IHC / FISH) larvae overnight at room temperature in TOTO-3 iodide or SYTOX Green working solution. - Wash larvae 3×5 min in PBST. - Transfer larvae to 80% glycerol in PBS and image as soon as possible. **IV. Mounting** Before mounting, larvae should have spent at least six hours in 80% glycerol in PBS, in order to be completely equilibrated. Melt mounting media in water bath and maintain liquid in 40 degree Celsius heating block or water bath. Larvae are mounted in a sandwich of one large cover slip (24×60 mm; used as “slide”) and a small coverslip (18×18 mm), with medium sized coverslips (22×22 mm) used as spacers. The sandwich is prepared by gluing with cyanoacrylate glue two stacks of small cover slips on one large cover slip at about 8 – 10 mm distance between the two stacks (Fig. 1). In general, three spacer cover slips (total thickness of 3×160 micrometer) are sufficient for two to four day old larvae. The spacers should generate a space thicker than the larvae to be mounted in order to avoid squeezing of the fixed tissue, which can cause deformations or damaged / torn tissue. 1. Put a single larva in an hourglass with some 80% glycerol in PBS. - With two hollow needles, remove the yolk and cut off the tail (helps to keep the larva in place when mounted). - Transfer larva with the tip of a hollow needle to a cover slip (24×60 mm) prepared with cover-slip spacer stacks. - Fill the area between the spacers with liquid warm mounting medium. - With the help of the hollow needles, orient the larva into the right position. - Place a small cover slip (18×18 mm) on the spacers and confirm that the embryo is not shifted using a dissecting microscope. The agarose solidifies. - Fix the chamber by applying spots of nail polish (Fig. 1). - Incubate mounted larva overnight in the dark in a humid chamber at room temperature to let it equilibrate (This is very important and can significantly improve the quality of the TOTO-3 nuclear stain as it might also equilibrate the nuclear staining.) **V. Microscope setup and confocal imaging** 1. For imaging place the cover slip sandwich with the mounted larva into the custom made aluminum frame (Fig. 2). - Fix the cover slip sandwich in the aluminum frame with modeling clay (Fig. 2). (This set up allows easy handling of the sample when it comes to turning around the cover slip to record a stack from the opposing side, here the ventral side). - Mount the aluminum frame on the microscope stage. - Perform recording of stacks in the first scan position (dorsal anterior part of the head); for high dynamic range imaging, record two stacks at different laser intensities; optimize laser for first stack such that all signal is in linear range, and for second stack such that signal deep in the brain is best. The depth in z-direction should be sufficient to cover most of the ventral brain. - Perform recording of stacks in the second scan position (dorsal posterior part of the head). The imaged volume has to overlap with the first position by about 20% to enable correct stitching. Same z-stack depth and laser settings as for first position. - Manually turn the sample coverslip sandwich fixed in the aluminum fame upside-down to record the ventral side. - Perform recording of stacks in the third scan position (ventral anterior part of the head), same laser settings as for first position. - Perform recording of stacks in the fourth scan position (ventral rostral part of the head), same laser settings as for first position. High dynamic range is obtained by recording each staining, the specific stain as well as the nuclear stain, with two intensities for every side (dorsal and ventral) and part (frontal and rostral) scanned, ending up with four channels per scan. The two intensities are individually adjusted once per larva with the first scan and kept equal for all the following scans. The low intensity is recorded first to minimize bleaching and is set in a way that no overexposure occurred, whereas the high intensity is set in a way that structures deep in the brain are fairly visible independent of how strongly the surface structures are overexposed. Microscope settings - Microscope: Zeiss LSM510-i-NLO laser scanning confocal microscope - Objective: LD LCI Plan-Apo 25x/0,8 Imm Korr multi immersion objective - Stack size: 512×512 pixels; 1 cubic micrometer voxel - Zoom factor: 0.7 - Immersion medium setting of objective: glycerol - Immersion medium: glycerol - Scan mode: 12bits, multi track - Lasers, filters, excitation and emission wavelengths: See Figure 3 **VI. Multiview resonstruction, stitching, and attenuation correction** The recorded stacks are further processed using the ViBE-Z software package through a web interface (http://vibez.informatik.uni-freiburg.de/). 1. Recorded stacks are imported into ImageJ (http://rsbweb.nih.gov/ij/index.html) using the import plugin appropriate for the confocal microscope brand type software. - Install the HDF5 data format plugin in ImageJ (http://lmb.informatik.uni-freiburg.de/resources/opensource/imagej_plugins/hdf5.html). - Save stacks in HDF5 format. - Create account at “http://vibez.informatik.uni-freiburg.de/”. Upload files and follow instructions for processing at this site. ### Timing - Day 1 cross fish - Day 2 collect eggs and sort embryos - Day 3 larvae are 24 hours old - Day 4 larvae are 48 hours old - Day 5 larvae are 72 hours old, fix embryos over night (most recordings were done with 72 hours old larvae) - Day 6 stop fixation, dehydrate, wait at least one hour before rehydration (better: start on the next day) - Days 7 – 9 in situ staining or immunostaining - Day 9 nuclear staining - Day 10 stop nuclear staining, larvae into glycerol, wait six hours and mount - Day 11 record larvae (approximately four hours per larva) - Day 12 data processing ### Troubleshooting - Weak FISH or IHC staining: - Embryos should be fixed as freshly as possible. - The proteinase K digest is a crucial step in both IIA and B protocols and may always be a step worth to be optimized if problems occur, especially for older larvae. - Always make sure, all solutions are carefully prepared and probes / antibodies are working. - High background in WISH - Can be reduced by optimizing the probe concentration as well as with the hybridization water bath temperature (higher temperature lead to a more stringent environment and might therefore lower the background). - High background in the IHC - Might be decreased by reducing the antibody concentration. - A weak or uneven nuclear staining - Can be caused by a short staining time. It is important, especially in older larvae, that the staining is performed overnight, as the stain needs to diffuse evenly deep into tissue. It turned out that a longer staining time also enhances the signal to noise ratio (lower apparent background), as there is less stain visible in the cytoplasm and the nuclei become more clearly visible. This may be caused by TOTO3 binding equilibrium to DNA versus double stranded RNA. The nuclear stain however also diffuses over time out of the samples, thus it is suggested to do the imaging as soon as possible after the stain is complete. - It is not recommended to increase the concentration of the staining working solutions, as this increases the background rather than improving the staining quality. - Bleaching - If too much bleaching occurs during imaging, the staining may be too weak and a fresh staining might be necessary. Bleaching may be reduced if multiple imaging is avoided. Recording the low channels first is also highly recommended. It might also be helpful to check that the intensities are as low as possible. - Avoid optimizing recording settings on the same embryo that you take stacks, which may cause bleached planes in the stack data. Usually within an identically treated batch larvae are fairly similar in stain intensity, and some larvae should be sacrificed to find the optimized scan setting, which are then applied to a fresh larva. - Orientation of larva in mounting medium - Images of zebrafish are typically displayed thus that anterior points to the left and posterior to the right. It can be helpful to mount a larva in a way that it will automatically be recorded pointing into the right direction. This depends on the microscope setup as well as on the microscope software. Thus, depending on these two factors, the axis along which a larva is mounted as well as the direction can vary. - Problems with data processing using the ViBE-Z web-interface - It is important for the registration that embryos are not damaged, deformed or squeezed. To avoid damaged tissue, embryos should be as freshly fixed as possible, because long storage times can lead to artifacts in the tissue. Staining and mounting should be performed as gently as possible. If any squeezing occurs, use more spacer cover slips for mounting. For the correct stitching of the dorsal and ventral side data stacks, it is important to scan as far through the sample as possible, optimally through the whole head from each side. In case the data should also be registered to the anatomical standard reference larvae, it is important to record both the anterior and posterior regions of the brain, including the anterior tip of the notochord and otic vesicles, as these landmarks are used for registration of data. ### References 1. Emmenlauer, M. et al. XuvTools: free, fast and reliable stitching of large 3D datasets. *J Microsc* 233, 42-60 (2009). - Filippi, A. et al. Expression and function of nr4a2, lmx1b, and pitx3 in zebrafish dopaminergic and noradrenergic neuronal development. *BMC Dev Biol* 7, 135 (2007). - Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. *Nat Methods* 2, 932-940 (2005). - Holzschuh, J. et al. Noradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid and require tfap2a for expression of the neurotransmitter phenotype. *Development* 130, 5741-5754 (2003). - Huisken, J., Swoger, J., Del, B.F., Wittbrodt, J., & Stelzer, E.H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. *Science* 305, 1007-1009 (2004). - O. Ronneberger, K. Liu, M. Rath, D. Rueß, T. Mueller, H. Skibbe, B. Drayer, T. Schmidt, A. Filippi, R. Nitschke, T. Brox, H. Burkhardt, and W. Driever. ViBE-Z: A Framework for 3D Virtual Colocalization Analysis in Zebrafish Larval Brains. *Nature Methods* (online June 17, 2012). - Westerfield, M., *The Zebrafish Book: A Guide to the laboratory Use of Zebrafish*, 4th ed. (University of Oregon Institute of Neurosciences, Eugene OR, 2000). ### Acknowledgements We would like to thank the Life imaging center for support and help with the confocal microscopes and S. Götter for excellent fish care. This work was funded by the Excellence Initiative of the German Federal and State Governments (Centre for Biological Signalling Studies EXC 294; Freiburg Institute for Advanced Studies), as well as by the EC projects 223744 (ZF-HEALTH) ### Figures **Figure 1: Mounting in coverslip sandwich**  *Mounting scheme using the coverslip sandwich. (A) shows a side view and (B) a top view of how a larva is mounted*. **Figure 2: Setup of sample holder on microscope stage**  *Setup of sandwich coverslip on microscope stage. To enable turning the sample upside-down, the cover slip with the embedded larva is placed into an aluminum frame and fixed with modeling clay. The frame mounted coverslip sandwich is set up on the microscope stage*. **Figure 3: Laser and filter settings**  *Table of lasers, filters, excitation and emission wavelengths used for the different fluorophores*. ### Associated Publications **ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains**. Olaf Ronneberger, Kun Liu, Meta Rath, Dominik Rueβ, Thomas Mueller, Henrik Skibbe, Benjamin Drayer, Thorsten Schmidt, Alida Filippi, Roland Nitschke, Thomas Brox, Hans Burkhardt, and Wolfgang Driever *Nature Methods* [doi:10.1038/nmeth.2076](http://dx.doi.org/10.1038/nmeth.2076) ### Author information **Meta Rath, Alida Filippi & Wolfgang Driever**, Driever Lab, University of Freiburg **Roland Nitschke**, Life Imaging Center LIC, University of Freiburg **Olaf Ronneberger**, Informatics Institute, University of Freiburg Correspondence to: Wolfgang Driever (driever@biologie.uni-freiburg.de) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2408) (2012) doi:10.1038/protex.2012.031. Originally published online 22 June 2012*.

Bones are one of the oldest materials used to create musical instruments. Currently, the world’s oldest known instruments are flutes made out of bones (Turk, Turk, and Otte 11). In fact, bones have been used to create or enhance musical instruments in a variety of settings throughout history and in modern day instrument making. Bone bull roarers, jaw bone percussion, clappers, trumpets, drum shells, lyres, or construction parts, such as frets, plectrums, pipes and pipe fittings, embouchure adjustments, or percussive strikes are just a few of the more common uses of bones in musical instrument construction. One man even made a guitar out of the skeleton of his dead uncle to memorialise the person who influenced his musical tastes and career (Bienstock). Bones can therefore be taken as a somewhat common material for making musical instruments. All of these instruments share a common trait, and not just the obvious one that they are all made out of or incorporate bones. None of these instruments are intended to represent something monstrous. Instead, they represent the ephemeral nature of humanity (Cupchik 33), a celebration of lineage or religious beliefs (Davis), or simply are the materials available or suitable to create a sound-making device (Regan). It is not possible to know the full intentions of a maker, in many cases, but a link to monstrosity and a representation of the ‘horrific’ or ‘freakish’ seems missing for the most. There are instruments, however, that do house this sentiment and some that utilise bones in the construction with the purpose of making this connection between the remains and something beast-like. In this article, I argue that the Bone Guitar Thing (BGT) built and played by raxil4 is one of those instruments. Introducing the 'Thing' Raxil4 is the stage name of sonic artist Andrew Page. He has been playing his Bone Guitar Thing for almost twenty years in a variety of settings (Page, email interview, 25 June 2021). The instrument has undergone slight changes during that time, but primarily it has retained its specific visual, timbral, and underlying associative features. The BGT is complex, more so than it may seem at first. By investigating the materials used, the performance techniques employed, and raxil4’s intentions as a musician, instrument maker, and community member within his circles of activity, the monstrous nature of the BGT comes to light. The resultant series of entanglements exhibits and supports a definition of what is a 'monster' that, like several definitions in monster theory discourse (Levina and Bui 6; Cohen 7; Mittman 51), includes challenging that which may be seen as ‘normal’ and thereby may nurture levels of unease or fear. However, in the case of the BGT, that which is monstrous is simultaneously being taken as something positive alongside its beast-like characteristics, and rather than evolving into something that needs to be repressed or eliminated, the ’monster’ here becomes a hero or champion, colleague, or even a friend. The Bone Guitar Thing is not really a guitar. It is a zither with a piece of driftwood for a base, (currently) five strings, and an electric pick-up (see Fig. 1). The bridge for the instrument is two bones, and the pitch and timbre of the strings is sometimes changed with bones used for Cage-like preparation (Cage 7-8; Bunger). Bones are also used to play the instrument, sometimes like a plectrum, others like a hammered dulcimer, or occasionally, simply pounding the string or the soundboard with great force to make a combination of percussive and string sounds. Glissandos are created by using the plectrum bones as a slide, and Page also uses jaw bones to introduce ratchet sounds, string scraping, and precise pitch bending (with the sharper edged part of the bones) (raxil4, “Livestream”). The instrument is electric, so the bones are enhanced with guitar pedals (typically reverb, distortion, and octave-splitter; Page, email interview, 25 June 2021), but the tonal qualities retain a semblance of the bone usage. Fig. 1: raxil4's Bone Guitar Thing. Photograph: Andrew Page. Page often uses the BGT as part of his sonic arsenal to perform dark ambient music, noisescapes, improv music, or live film soundtracks both in live concerts and recording situations. He plays solo as much as with ensembles, and more often improvises his music or parts, but occasionally works with predetermined organisation or scores of some description (although he admits to typically abandoning predetermined passages or scores during live performances; Page, email interview, 14 July 2021). Currently in London, raxil4 presents concerts in a variety of settings, typically well-suited for his brand of sonic art, such as Ryan Jordan’s long-running concert series Noise=Noise (raxil4 feat. King Sara), experimental music shows at the Barbican (raxil4 + King Sara + P23), and dark ambient showcases promoted and arranged by one of his record labels, Sombre Soniks (Wright). Sounds beyond Words: Monstrous Music One series of performances in which raxil4 used the BGT took the form of an immersive theatre show produced by Dread Falls Theatre called Father Dagon, based on the works of horror author H. P. Lovecraft. The performance incorporated a breaking of the ’fourth wall’ in which the audience wanders freely through the performance space, with actor- and sometimes audience-interactive musical performances of partially improvised, partially composed passages by musicians located throughout the set. Director and writer Victoria Snaith considered the use of live, semi-mobile, experimental music dispersed through the audience (mixed with an overall backing soundtrack) as heightening the intensity of the experience by introducing unfamiliar aspects to the setting. She discusses having made this decision based on Lovecraft’s own approach to story-telling that highlights a sense of unfamiliarity and therefore sense of “fear of the unknown”. The usefulness of creating unfamiliarity in this context can serve to support the parts of the narrative that contains supernatural and monstrous aspects. Given that the elements of the supernatural and horrible monsters in Lovecraftian tales are primarily indescribable (both because Lovecraft would recount beasts and fantastic magical happenings in his works as being such, and because in a practical theatrical situation, these things would be impossible to describe, especially without text or specific props or costumes, which the show purposefully uses sparingly, also as a conscious choice to embrace the unknown). Sounds created on instruments that are unique, or generated through unusual performance techniques would lend themselves to being more difficult to describe, and therefore fitting to support a desire to present something regarded as also difficult to describe, that being supernatural happenings or horrific creatures. (Connor 77) Page’s use of the BGT in these performances added directly to this notion both sonically and visually. The homemade nature of his instrument increased the potential that audience members would be less familiar with the source of his sounds, even if they were watching him perform, and the resultant soundscape he provided introduced harsh timbres, undulating pads, and aggressive punctuation of movement. Page sees the BGT as an instrument “reclaimed from the watery depths” (matching the theme of the show’s narrative), therefore as one fitting into the Lovecraft show “quite nicely” (Page, email interview, 25 June 2021). He likens the sounds created by the BGT as presenting “otherworldly melodies” akin to those played by Erich Zann (a character in another Lovecraft story who conjures a gateway to an alternate dimension full of indescribable creatures and nightmares via performing unusual music on his viola de gamba), which Page also sees as fitting (ibid.). His instrument in this setting as a producer and provider of unfamiliarity is supportive of constructing and maintaining a definition of “monstrous” or “terrifying” (Levina and Bui 6). Fig. 2: raxil4 performing in Dread Falls Theatre's Father Dagon, London 2012. Photograph: Pierre Ketteridge. Finding Community in the 'Freakish' Raxil4 also notes that the Bone Guitar Thing is appropriate for creative input within improv music circles (Page, email interview, 25 June 2021). Generally speaking, contemporary improv music (meaning the broad genre) is improvised performance focussing on sonic exploration over melodic, harmonic, or rhythmic content (even though all will be present in most cases; Toop 132-137). In my experience working with improv musicians since 1981, I find that these performers typically attempt to create sounds that are unusual or unexpected. Players often embrace extended techniques, repurposing non-musical items to be sound-making devices, and employ self-built instruments. Improv musicians seek to break free from the constraints of what may be seen as Western standard musical practices (ibid.), but they simultaneously strive to uphold some parallel aspects of artisanship and virtuosity, perhaps as a means to validate their departure from Classical/mainstream music norms. The instruments and approaches can be seen as factors that separate the experimental artists from the conservatory-based performers, yet still affords them the clout of being hard-working, innovative, expressive, and professional. As the name implies, improv music emphasises improvisation. André Hodier (23-36) in his classic book The Worlds of Jazz likens improvising jazz musicians to an alien race who battle each other on a daily basis (via jazz battles) in order to see who resides at the top of the improvisation chain. Improv musicians (some of whom come from a jazz background) tend to engage in this sort of hierarchical status ranking system using a much more ’polite’ and co-supportive mentality (at least in the scenes in which I have been privileged to participate). Improv musicians can occasionally embrace a friendly attitude that one should surpass the experimental nature of other performers, and may do so by presenting a new sound, technique, or instrument. The BGT can serve this function. It can stand out among other improv musicians’ gear, even if a majority of the instruments are self-built, through its use of bones and its intentional evocation of something horrific. Improvised music is sometimes looked down upon by musical communities who value conservatory training, popular music, or more traditional Western classical approaches to music. Referring to avant-garde jazz in the 60s and 70s, Valerie Wilmer (6) recounts that critics and Classical music enthusiasts perceived experimental and improv music as “‘freakish’ and only worthy of passing interest”. The dynamic is different today, but the overall attitude remains, at least in part. The improv music scene is creatively valid, but in comparison to conservative or more mainstream music, incorporates more experimental practices, therefore sometimes musical form, interactions, and preparation is less obvious to audience members outside the experimental music circles. The Bone Guitar Thing also plays into this construction. It is artistically valid, yet perhaps simultaneously challenging to the less-experienced listener. The BGT in this setting is multifunctional. Page (email interview, 25 June 2021) sees the BGT as a means to cut through or rise above other improv musicians, partly by being more recognisable as a “freakish” instrument at performances where the music is already considered freakish by some outsiders. Additionally, the fact that Page has taken the time to make this instrument, and uses notably practiced techniques to create the sounds he introduces, may position him as an innovative professional, rather than a non-trained imposter. The BGT can (at least for raxil4, but for others as well) become a monster among monsters that allows Page to validate his brand of creativity (Ibid). Musical ’freakishness’ appears in other settings as well. An example of this is a performance in which raxil4 took part where an ensemble provided experimental music for a live tattooing event (raxil4, “Listening”). Here, the congruency with being monstrous or freakish is perhaps more overt. Similar to the soundscape being performed, Fenske (6) points out that tattoos may still be seen as unfit or unexpected for certain classes, genders, or education levels, and may even still be associated with illustrated circus performers of the past. Furthermore, Kinzey (32) suggests that avant-garde and counter-culture communities (such as ones where tattooing and live music converge in a single event) often value uniqueness that serves to “erase boundaries between everyday life and art”. The combined performativity of live music and tattoo inking (both the artistic activity and the art itself) associates raxil4 and the BGT with this non-mainstream circle (to some degree), potentially conjuring an identity of something freakish or monstrous to people with different values. Engaging with Expressive Objects The conception and evolution of the Bone Guitar Thing has its roots in personal experience, art experimentation, and material culture related to Page’s life and the musical communities in which he played and plays. In the past, Page endeavored to make small sculptures to be given as Christmas and birthday gifts from materials he found on the shore of the River Thames, many including bones. Page then began to create new musical instruments with what he had available. Page’s brother is a doctor specialising in gunshot wounds and knife trauma, and his apartment was filled with remnants of his brother’s occupation, including a number of crutches. From these, Page crafted his first instruments in this period: crutch harps that utilised the leftover medical devices to build stringed sound generators. He claims the instruments at first were not overly successful, so he began to experiment with his bone sculptures to create more serviceable instruments. An early attempt was a percussion instrument made from various found bones, which Page deemed the “Xylobone” (see Fig. 4). This instrument and advanced crutch harps (6-string tenor (see Fig. 3.) and 2-string bass) became his first arsenal of sound makers, but Page felt the instruments ultimately failed to meet expectations and opted to rethink his approaches and designs. Fig. 3: One of Page's 6-stringed crutch harps. Photograph: Andrew Page. Fig. 4: The Xylobone - raxil4's bone xylophone percussion instrument. Photograph: Andrew Page. The BGT was intended to be more “playable”, “expressive”, and audible to battle louder co-performers. As mentioned, the driftwood base and bones for the instrument originated from the River Thames. The electronics come from a destroyed guitar that was the result of performing in a previous project in which Page was the singer, where the guitarist “had a habit of smashing his guitars on stage, in a sort of expensive tribute to [grunge guitarist] Kurt Cobain" (Page, email interview, 25 June 2021). The BGT started off as a 6-string zither that used guitar-gauge steel strings, but according to Page, given the harsh performance technique of beating or scraping the strings with bones, he was encouraged to switch to using wound, bass-gauge strings, affording him a lower pitch and greater resistance to energetic performance practices. One tuning peg, however, snapped off quite early in its life (as it was in a thinner, more weathered part of the driftwood), leaving the instrument one string shorter. Page says he likes to think that the instrument decided itself that it would be a “5-stringed beast” (ibid.). Conclusion The Bone Guitar Thing is, in fact, beast-like, at least in the settings, sonic attributes, and mindsets of the player and the communities in which the instrument is played, but it may not be the case that this beast-like nature is equal to being monstrous. Cohen (3-25) in his discussion of seven potential monster theories outlines several different notions of what can be considered “monstrous” and relates the monster in each theoretical situation to those fearing the monstrous construct. Most closely related to the situation in which the BGT is observed is a parallel theory based on the concept of “Us versus Them”, meaning “Us” as those who are dealing with the monster in question, and “Them” as being those on the side of the monster or the monster itself (Cohen 19-20). However, with the BGT, the monster is not unanimous with “them”, but rather with “us”. In all the situations outlined here, the instrument takes on the role of a beast, but not a negative role for Page (email interview, 14 July 2021) or fans of raxil4 (Wright). Instead, the beast is more like part of the team of noise makers actively engaged in the community’s activities of creation, entertainment, identity, and validation of values upheld thereof. Each of the performance settings can be argued to exhibit a sense of welcoming outsiders or praising diversity, rather than ostracising it. The Lovecraft performance and story were constructed on the premise of questioning what is a monster and who determines that definition. The Bone Guitar Thing supports and interacts precisely within this parameter to enhance the artistic commentary presented. Within the improv music setting, the instrument assists Page to achieve uniqueness among that which is already unique and highlights the values of community including a show of innovation, exploration, and personal performance technique development. For the live tattooing, the instrument stands out as a unifying sonic flag, connecting other (perhaps less-monstrous) artists into a stronger group of alternative creatives. Effectively, the BGT is a 'freak among freaks', serving to simultaneously fit in and rise above, all while maintaining a sense of “us” within respective circles. The beast-like nature is not entirely an outward force. Page (email interviews, 25 June 2021 and 14 July 2021) is aware that he has received no formal education in music. He admits he is less familiar with music theory, and more familiar with the science and technology behind the music. Page considers himself to be experimental in his approach to sound creation, which he sees as being more unique due to ignoring the “rulebooks” (ibid.). As a result, he feels (at least a slight) pressure of feeling “unprofessional” or “correct” in the eyes of Western conservatory-trained musicians and composers or those with a similar mentality (Page, email interview, 25 June 2021). The BGT was also, to a degree, built to battle being told what was “right”. For Page, his instrument is akin to a beast that helped him break free of the constraints of Western tonal and virtuosic constraints. “I made my own [instrument] so that nobody could tell me I was playing it the wrong way” (ibid.). His “beast” helped him break down barriers and asserted himself as an innovative musician and creative professional. So, then, the Bone Guitar Thing is a monster; sonically, visually, and physically. It represents a monster, it is called “the beast”, and it takes on the role of a terrifying creature raging through (sometimes, extremely quietly – raxil4; raxil 4 feat. King Sara; raxil4 + King Sara + P23) soundscapes, settings, and performances, rallying the like-minded and routing the unsuspecting or “others”. That is an overdramatic take on the situation, perhaps, but the instrument does uphold a series of values and creative aesthetics that fosters positive relationships between the artist, the community, and the sonic and physical qualities of the zither. Rather than being a device that places a horrific barrier to be overcome in an “us versus them” scenario, the monster takes on an alternate role and becomes a source of empowerment for “outsiders” or marginalised groups or people (Mittman 51). Thus the Bone Guitar Thing allows Page to demolish barriers and amalgamate fellow community members into a larger version of “us” to create a space in which the beast is no longer a monster. References Bienstock, Richard. “Man Builds Guitar Out of His Dead Uncle’s Skeleton.” Guitar World 11 Feb. 2021. Web. 13 June 2021 <https://www.guitarworld.com/news/man-builds-guitar-out-of-his-dead-uncles-skeleton-uses-it-to-play-black-metal>. Bunger, Richard. The Well-Prepared Piano. Colorado Springs: Colorado College Music P, 1973. Cage, John. Empty Words: Writings ’73-’78. Middletown, CT: Wesleyan University P, 1981. Cohen, Jeffrey Jerome. “Monster Culture (Seven Theses).” Monster Theory: Reading Culture. Minneapolis: U of Minnesota P, 1996. 3–25. Connor, Will. “Performing the Sounds of Darkness: An Exploratory Discussion of Musical Instruments and the Gothic Aesthetic.” The Dark Arts Journal: Reimaging the Gothic 2.I2 (Autumn 2016). 26 June 2021 <https://thedarkartsjournal.files.wordpress.com/2017/04/the-dark-arts-journal-2-21.pdf>. Cupchik, Jeffrey. “Buddhism as Performing Art: Visualizing Music in the Tibetan Sacred Ritual Music Liturgies.” Yale Journal of Music & Religion 1.1 (2015): 31–62. Davis, Josh. “Some Bronze Age Britons Turned the Bones of Dead Relatives into Musical Instruments.” Natural History Museum. 1 Sep. 2020. 23 June 2021 <https://www.nhm.ac.uk/discover/news/2020/september/bronze-age-britons-turned-the-bones-of-dead-relatives-into-musical-instruments.html>. Fenske, Mindy. Tattoos in American Visual Culture. New York: Palgrave, 2007. Hodier, André. The Worlds of Jazz. New York: Grove P, 1972. Kinzey, Jake. The Sacred and the Profane: An Investigation of Hipsters. Winchester, U.K.: Zero Books, 2012. Levina, Marina, and Diem-My T. Bui. “Introduction: Toward a Comprehensive Monster Theory in the 21st Century.” Monster Culture in the 21st Century: A Reader. Eds. Marina Levina and Diem-My T. Bui. New Delhi: Bloomsbury. 1–14. Mittman, Asa Simon. “Introduction: The Impact of Monsters and Monster Studies.” The Ashgate Research Companion to Monsters and the Monstrous. Eds. Asa Simon Mittman and Peter J. Dendle. London and New York: Routledge, 2013. 44–60. Raxil4. Listening Circuits: 19/06/21 with Live Tattooing from Catmouse. 21 June 2021. 23 June 2021 <https://www.youtube.com/watch?v=VZgUC5TTOxk&list=LL&index=3>. ———. raxil4 – Livestream for Iklecktik: 21/06/20. 22 June 2020. 23 June 2021 <https://www.youtube.com/watch?v=-zW-Mw2jRDQ&list=LL&index=6>. Raxil4 feat: King Sara. raxil4 feat: King Sara – Sawbones 13 – Live @ Noise=Noise (14/01/13). 26 Jan. 2013. 23 June 2021 <https://www.youtube.com/watch?v=fxFMA77yQ_A&list=LL&index=5>. raxil4 + King Sara + P23. raxil4 + King Sara + P23 – Barbican: 15/08/13. 11 Sep. 2018. 23 June 2021 <https://www.youtube.com/watch?v=N619ooZxx-0&list=LL&index=4>. Page, Andrew. Email interview. 25 June 2021. ———. Email interview. 14 July 2021. Regan, Marty. Video interview. 13 July 2021. Snaith, Victoria. Personal interview. 17 April 2016. Toop, David. Ocean of Sound. London: Serpent’s Tail, 2001. Turk, Matija, Ivan Turk, and Marcel Otte. “The Neanderthal Musical Instrument from Divje Babe I Cave (Slovenia): A Critical Review of the Discussion.” Applied Sciences 10-1226.2 (2020): 1–11. Wilmer, Valerie. As Serious as Your Life. London: Serpent’s Tail, 2018. Wright, Kevin. Email interview. 29 June 2021.

Authors: Yeh-Chuin Poh & Ning Wang ### Abstract Mechanical forces are known to play a significant role in biological processes. These forces can be transmitted to the cell through the cytoskeletal filament network, inducing different biochemical responses within the cytoplasm. Although there have been ample reports showing that cytoplasmic enzymes can be directly activated by a local stress on the cell surface via integrins, there has been no evidence that mechanical forces can directly alter nuclear functions without intermediate biochemical cascades. Recently, we showed evidence that forces on the cell membrane can be transmitted directly into the nucleus, inducing structural changes of protein complexes in Cajal bodies. Here, we describe a protocol that utilizes the optical magnetic twisting cytometry (MTC) for force application and fluorescent resonance energy transfer (FRET) to monitor the dynamics and interaction of various Cajal body proteins. ### Introduction It is well known that human bodies are constantly under the influence of mechanical forces. These mechanical forces influence the growth of tissues and organs. Cells integrate both chemical and mechanical cues to regulate biological processes as diverse as differentiation, vascular development, tumor growth and malignancy (1-5). However, little is known about the mechanism by which individual cells sense the mechanical forces and convert them in to biochemical signals within the cell and influence the gene expression, a process known as mechanotransduction. Advances in the field of mechanotransduction have demonstrated that focal adhesion complex proteins such as spectrin (6), talin (7), and integrin (8) can be deformed, unfolded, and thus activated by forces of physiologic magnitudes. Proteins and enzymes within the cytoplasm can be rapidly activated and phosphorylated upon mechanical stress (9, 10). Stem cells differentiate in respond to different surface topology (11), substrate rigidity (12), and applied stress (13), a clear indication of gene expression change within the nucleus. Nonetheless, direct force-altered protein localization/activity and thus gene expression within the nucleus remains elusive. Immunoblotting and immunostaining have been used to study mechanotransduction (14), but these techniques do not provide sufficient resolution and real time results in a single living cell. Because gene expression in a live cell involves many complicated processes in the cytoplasm and the nucleus and takes time, it is impossible, at the present time, to attribute any changes in gene expression to direct effects of the applied force at the cell surface, without involving intermediate biochemical signaling cascades. Therefore, we asked if localizations of protein complexes in the nucleus can be directly altered by a local surface force, since there is evidence that interactions among nuclear proteins are critical in regulating gene expression. We employed a FRET (fluorescence resonance energy transfer) based technique to monitor the dynamics and interaction of proteins within the Cajal Body (CB) complex in response to mechanical stress (15). CBs are critical for the biogenesis and recycling of several classes of small ribonucleoprotein (snRNP) complexes involved in pre-mRNA splicing and preribosomal RNA (pre-rRNA) processing (16, 17), and assembly and delivery of telomerase to telomeres (16-19). Recent advances in the dynamics, assembly, and function of CBs suggest that the CBs organize as a direct reflection of highly active genes with which they are physiologically associated (20). Intermolecular FRET can be used to visualize protein-protein interactions. In this protocol, we labeled various CB proteins with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). The CFP labeled protein acts as the donor while the YFP labeled protein act as the receptor. Only CFP is excited during the experiment while both CFP and YFP were monitored simultaneously. By observing the relative intensity changes of CFP and YFP, we can then quantify the distance and interaction between CB proteins. When the CFP and YFP labeled proteins are close to each another (<10nm), FRET occurs when the emission of CFP is transferred to excite YFP. As we observe the FRET ratio of CFP/YFP, there will be a decrease in FRET ratio when the two proteins come closer to each other, because there is a decrease in CFP intensity and an increase in YFP intensity (anti-correlation). On the other hand, when the two proteins are separated and thus there is an increase in distance between the two proteins, the ratio of CFP/YFP will increase. Here, we outline the use of magnetic twisting cytometry (MTC)21 in detail to study the spatial and temporal mechano-chemical response within the cell nucleus. We also describe the use of a dynamic sinusoidal load to quantify the viscoelastic and dissipative behavior between protein pairs within the CB. Our results showed that mechanical force at the apical membrane can be directly transmitted through the actin microfilaments and nuclear envelope to remote cites within the nucleus. The stress induced protein dissociation was rapid and did not require intermediate biochemical signaling, diffusion, or translocation. ### Reagents 1. CFP and YFP plasmid probes of coilin, SMN, fibrillarin, Nopp140, WRAP53, SART3, SmE, SmG (from Miroslav Dundr, Rosalind Franklin University of Medicine and Science, North Chicago) - mCherry-Lamin A plasmid (from Peter Kalab, National Institute of Health) - HeLa cells (ATCC) - Lamin A/C -/- (LMNA-knockout) mouse embryonic fibroblast (from Colin Stewart, - National University of Singapore, Singapore) - Plectin -/- mouse fibroblast (from Gerhard Wiche, University of Vienna, Germany) - CO2-independent medium (Invitrogen) - Collagen, type I from rat tail (solution, Sigma, 091M7675) - Ferromagnetic beads (Fe3O4; 4.5-µm diameter) (from W. Moller, Gauting, Germany or J. Fredberg, Boston, MA; magnetic beads with various surface properties are commercially available in an assortment of sizes from Spherotech, Inc., Lake Forest, IL) - Dimethyl sulfoxide, sterile-filtered (DMSO; Sigma) - Fetal bovine serum (FBS; HyClone) - Opti-MEM I medium (Invitrogen) - Penicillin-streptomycin (Invitrogen) - Phosphate-buffered saline (PBS; HyClone) - L-Glutamine (100x) (Invitrogen) - Lipofectamine 2000 (Invitrogen) - Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma) - 2% Bis solution (Bio-Rad Laboratories, 161-0142) - 40% Acrylamide solution (Bio-Rad Laboratories, 161-0140) - 3-aminopropyltrimethoxysilane (Aldrich, Product # 281778) - 0.5% gluteraldehyde (Sigma-Aldrich, Product # G6257) - 10% Ammonium persulfate (APS, Bio-Rad) - TEMED (Bio-Rad) - HEPES (Sigma) - Sulfo-SANPAH (Pierce, Product # 22589) - 0.2μm Florescent latex beads (Molecular Probes) - 0.05M Carbonate buffer (pH 9.4; bioWORLD) - Glass-bottomed culture dishes (35-mm diameter; Cell E&G;) - 12 mm circular cover glasses (Fisher, cat. # 12-545-80) - Synthetic RGD-containing peptide [Ac-G(dR)GDSPASSKGGGGS(dR)LLLLLL(dR)-NH2, - Peptide-2000 (Telios) (18); Peptides International, Inc., Louisville, KY] ### Equipment 1. 40×0.55 numerical aperture (N.A.) air and 63×1.32 N.A. oil-immersion objectives (Leica) - CCD camera (Hamamatsu; model C4742-95-12ERG) - CFP/YFP Dual EX/EM (FRET) Filter sets for FRET experiments (Optical Insights): CFP: excitation S430/25, emission S470/30; YFP: excitation S500/20, emission S535/30. The emission filter set uses a 505-nm dichroic mirror. - Dual-View imaging system (Optical Insights) - Inverted microscope (Leica, DMIRE2) - Matlab (Mathworks) - MTC device (Commercially available via special order from EOL Eberhard, Obervil, Switzerland) ### Procedure **Coating magnetic beads with RGD** 1. Suspend the magnetic beads stock in 95% alcohol (for sterilization) and aliquot them into small 2ml vial each containing 1mg of beads. - Leave a vial open and evaporate the alcohol out. - Add 1.5ml of PBS buffer to rinse the beads. Centrifuge it down and discard the PBS carefully. - Add 1ml of Cabonate buffer to the beads. - Add 50 μg of RGD peptides (diluted in DMSO) to the bead-buffer solution. - Rotate the beads at 4OC overnight. - Before using beads, centrifuge the beads and discard the supernatant RGD. Then rinse it once with PBS as described in step 3. - Store the coated beads in serum free DMEM. **Cell culture and transfection** 1. All cells used for experiments are preferred low passages. - Regular cells culture was done in T-25 flask and maintained in DMEM supplemented with 10% FBS100U/ml penicillin, 100μg/ml streptomycin, and 2mM L-Glutamine at 37oC in 5% CO2. - For experiments, cells need to be prepared 3 days in advance. Day 1, coat the 35mm glass bottom dishes with Collagen-I and store at 4oC to allow absorption of collagen on to the glass surface. - Day 2, sterilize the collagen-coated culture dish by leaving it under UV light for 10 minutes. Then seed (~300,000) cells in the collagen-coated 35-mm glass bottom dish such that it is ~80% confluent the following day. - Day 3, double transfect the cells with plasmid constructs of CFP and YFP labeled proteins. Dilute 1μg of CFP plasmid and 1μg of YFP plasmid to 100μg of Opti-MEM I medium in a small vial. - In another vial, add 4μl of Lipofectamine 2000 to 100μg of Opti-MEM I medium in a small vial. - Wait 5 minutes in room temperature before mixing the contents of both vials together. Then wait another 20 minutes. - Add the total ~200μl of DNA-Lipofectamine mixture to the dish containing cells. - Optional: To minimize photobleaching, 0.05mM Trolox solution was added to the dish along with the DNA-Lipofectamine mixture (22). - Incubate for 6 hours at 37oC in 5% CO2 before replacing the culture medium with regular DMEM culture medium. - Day 4, cells are transfected and ready for imaging. **Magnetic Twisting Cytometry (MTC)** 1. On the day of the experiment (day 4 in t in the previous section), take the dish out of the incubator and remove most of the culture medium, such that only the cells in the center well (glass region) is slightly covered in medium. - Add 20μl of RGD-coated magnetic beads (~20μg of beads) to the center well of the dish by scattering them all over. - Carefully place the beads back into the incubator and leave for 10 minutes to allow for integrin clustering and formation of focal adhesions surrounding the beads. - Remove cells from incubator and rinse it once with PBS. Avoid disturbing cells in the center well. Add and remove PBS gently by the side of the dish. - Add CO2-independent medium to the dish. This is to maintain the pH of the cell culture when it is exposed to the open while under the microscope. - Place the dish in the MTC stage where coils are located. Then place it on the inverted microscope. - Find a single cell that is well transfected with both CFP and YFP plasmids. The cell also needs to have a single bead attached to it. Exclude all cells that are not well transfected, have more than one bead attached, or are in contact with neighboring cells. - After the good cell is found, magnetize the magnetic beads by applying a strong magnetic pulse (~1000G, <0.5ms). - Now that the beads are polarized and magnetized, apply a magnetic field in the direction perpendicular to that of the magnetizing pulse. This will cause the bead to rotate. Input the parameter for MTC. Parameters of stress peak magnitude for FRET analysis are typically 17.5Pa (50G step load) or other magnitudes, where for phase lag analysis is 24.5Pa (70G oscillatory load). - While force is being applied, capture the necessary brightfield or fluorescence images. **FRET imaging and analysis** 1. For FRET imaging, the Dual-View imaging system was used to split the image into two (1344×512 pixels each). The top view filters for YFP, while the bottom view filters for CFP. Each image is 1344×1024 pixels and simultaneously captures both CFP and YFP activity. - While force is being applied by the MTC, FRET dual-view time course images are captured to monitor the protein-protein interaction within the nucleus before and after force. - After experiments are done and images obtained. A customized Matlab program is used to analyze the data. The program first divides the top (YFP) and bottom (CFP) image in to two separate files. - The region of interest (an individual CB in our case) is then selected. The program crops this region from the CFP and YFP images, then aligns them by cross-correlation. - A binary mask is then created for CFP and YFP images by using Matlab’s “graythresh” function. The binary mask is then multiplied with the fluorescent images generating images that have only the fluorescing region and a black background. - The CFP/YFP ratio value is then calculated for each individual pixel that has been aligned and cross-correlated. An average of the region is obtained and reported. Each image or time point will generate one CFP/YFP value. - Note: More details on the Matlab program has been described by Na S et. al. (14) **Polyacrylamide gels for traction force measurement** 1. Polyacrylamide (PA) gels with 0.2μm fluorescent beads embedded within are used to measure the traction force each cell generates. By varying the concentration of bis and acrylamide, different gel stiffness can be obtained. - To prepare PA gels, first smear 3-aminopropyltrimethoxysilane over the glass surface of a 35mm glass-bottom-dish using a cotton-tipped swab and let it sit there for 6 min. - Wash it thoroughly with water before applying 100 μl/ dish of 0.5% gluteraldehyde for 30 min. - Wash again thoroughly and let them dry. Avoid touching the glass surface throughout the whole gel making procedure. - Determine the bis:acrylamide solution proportions to get the desired substrate stiffness. 0.6, 2, and 8 kP, corresponds to 0.06% bisacrylamide and 3% acrylamide, 0.05% bisacrylamide and 5% acrylamide, 0.3% bisacrylamide and 5% acrylamide respectively. Prepare 1ml of each desired mixture in a small 2ml vial. - Add 10μl of 0.2μm fluorescent beads to the bis-acrylamide mixture. Before adding fluorescent beads, be sure to vortex or sonicate. - Add polymerizing activator/initiator to the beads-bis-acrylamide mixture. 10% APS at 1: 200 volume ratio (5μl in this case). TEMED at 1: 2000 volume ratio (0.5μl in this case). Mix everything together thoroughly. - Add 15μl of the mixture to the glass surface of the treated dish. (15 μl would give 75 μm thick substrates) - Flatten droplet with a 12mm circular cover glasses. - Turn the glass bottom dish upside down. This ensures the fluorescent beads to be closer to the top surface. - Place the upside down dishes in a 37oC incubator for 30-45 minutes. Elevated temperature helps in the polymerization. - After the gels are fully polymerized, flood the dish with 100 mM HEPES. Then carefully remove the circular cover glass with a single edge razor. - Make 1mM solution of SANPAH with DMSO and 100 mM HEPES. Add DMSO to SANPAH first to dissolve the solid powder, and then add it to HEPES. For example, 5mg SANPAH+50 μl DMSO+ 10 ml (100mM) HEPES. - Take out HEPES from the glass bottom dishes, dab excess HEPES with Kim wipes from around gel edge - Apply 200μl of SANPAH solution the gel (center well of dish). - Expose surface to UV for 6 min (6″ away from the lamp) to photo activate the gel surface. SANPAH color will turn dark. Without SANPAH treatment, collagen will not bind to gel surface. - Rinse off SANPAH with 100mM HEPES. - Repeat photo activation procedure once more and rinse it off with 100mM HEPES. - Coat the gel surface with the desired concentration of collagen and incubate at 4° C overnight. - Before seeding cells onto the gel surface, sterilize it under UV light for 10-15 minutes. - PA gels can be stored in PBS at 4° C for three weeks. - Note: To determine the ratio of bis to acrylamide for desired substrate stiffness, refer to references (23-25). **Traction Force Microscopy (TFM)** 1. Cells are cultured on the PA gels. Depending on the PA gel stiffness, the cell will generate different traction forces, and hence different magnitude of deformation. - Three images need to be captured. First is the brightfield or phase contrast image of the cell which will be used to identify the cell boundary. Second is the fluorescent beads marker image while the cell is still on the substrate. Third is the reference fluorescent beads marker image after the cell has been removed or trypsinized from the gel surface. - A customize Matlab program was used to analyze the traction force generated. The displacement field induced by each individual cell’s tractional forces was determined by comparing the fluorescent bead positions before and after trypsinization (cell-free and thus force-free). - An image correlation method where the flourecent images are divided into small window areas is used to determine the displacement vectors (26). - The root-mean-square (RMS) traction field was then calculated from the displacement field using Fourier Transform Traction Cytometry (FTTC) based on the Boussinesq solution (27). **Cell stiffness measurement** 1. The stress applied to the cell (in Pa) can be calculated from the applied twisting magnetic field (in G) by multiplying the bead constant (in Pa/G) with the applied twisting field (in G). The bead constant reflects the magnetic property of the bead and may differ from batch-to-batch. The beads are calibrated by immersing them in a known viscous fluid, and applying a constant magnetic field while measuring the remnant magnetic field (21). For example, a 50 G applies 17.5 Pa of stress to the cell if the bead constant is 0.35 Pa/G(9). - When a cell with a single bead bound to its apical surface is found under the microscope, an oscillatory stress of 0.3 Hz is applied using the MTC. - The MTC software tracks the displacement coordinates of the magnetic bead and saves them in a text file. - By quantifying the magnetic bead displacement, and the bead embedded area, the cell complex modulus can be estimated. A custom Matlab program is then used to calculate the cell stiffness. - The beads whose displacement waves are synchronized to the input sinusoidal signals were selected. This is to filter out spontaneous movements of the beads or microscope stage shifts. - Beads with displacements less than 5 nm (detectable resolution) and loosely bound beads were not selected for analysis. To increase the signal to noise ratio, the peak amplitude of the displacement was averaged over 5 consecutive cycles for each cell. - The complex stiffness is calculated using the equation G*=T/d. For each bead, the elastic stiffness G’ (the real part of G*) and the dissipative stiffness G” (the imaginary part of G*) was calculated based on the phase lag. The measured stiffness has the units of torque per unit bead volume per unit bead displacement (Pa/nm). - A finite element model is then used to convert the cell stiffness (Pa/nm) to modulus (Pa) based on the bead to cell surface contact (28)(Figure.1).  *Figure 1. Quantification of magnetic bead embedment in HeLa cells. An RGD-coated bead was bound to the apical surface of the cell for ~15 minutes before it was fixed and stained with phalloidin. Integrin-mediated focal adhesions form around the bead-cell contact area, giving rise to an actin ring. The bead embedment was estimated by measuring the actin ring diameter from the fluorescent image and comparing it to the bead diameter from the brightfield image (double arrows). Bead embedment in HeLa cells is 20-30%. Scale bar = 10 μm*. 9.More details on how to calculate cell stiffness have been described by Fabry B et. al. (29). **Phase lag quantification** 1. An oscillatory stress (0.3 Hz or 0.83 Hz) is applied to a cell that is well transfected, similar to the stress used to measure cell stiffness - Time course images of the bead, CFP labeled protein, and YFP labeled protein are captured while the cyclic force is being applied. - A custom Matlab program is used to analyze the images and the displacement of bead, CFP and YFP labeled proteins are determined. The phase lag of fluorescent proteins to the bead is then calculated. - One complete cycle of stress corresponds to 360o. For example, at 0.3 Hz, the period for one complete cycle is 3.33s. If CFP lags behind the bead displacement by 0.3s, that will correspond to a phase lag of ~32o. **Mean Square Displacement (MSD)** 1. For CB dynamics, an oscillatory stress (0.3 Hz) needs to be applied. Fluorescent images used for MSD analysis were obtained using single-view fluorescence filter. - Time course images of the bead, CFP labeled protein, and YFP labeled protein are captured before, during and after the cyclic force is being applied. - Binary images of the bead, CFP and YFP are obtained by using the “graythresh” Matlab function. The centroid coordinates of the bead and each fluorescing protein are then obtained. - The coordinates of each fluorescence particle obtained was then used to calculate the mean square displacement (MSD) of Coilin and SMN. The MSD before, during, and after mechanical loading were calculated using a customized Matlab program based (15). The same procedure is performed on bright-field images to obtain the bead MSD. ### Timing Preparation for experiments takes up to 3 days. Dishes need to be coated with collagen or other matrix proteins. Beads need to be coated with RGD or ligands to integrins. Cells need to be transfected. Depending on the transfection efficiency and how magnetic beads bind to the cell surface, locating an appropriate cell for data collection may take up sometime. The whole process of making PA gels may take a day (excluding incubation time of Collagen-I). ### Troubleshooting Cajal bodies are dynamic and spontaneously move. There are times when the observed CB moves out of focus, especially for those that are not tethered and exhibit simple diffusion. Intermolecular FRET is also difficult to observe because the stoichiometry of acceptors to donors can vary with transfection efficiencies29. On top of that, if the distance or orientations between the protein pairs are unfavorable, FRET may not be observed even if they both reside in the same CB complex. ### Anticipated Results One would expect to observe specific proteins in Cajal bodies that are tethered to chromatin and/or nucleoplasmic filaments to alter localizations or activities in response to a local surface force applied via integrins. ### References 1. Discher, D. E., Janmey, P. & Wang, Y. Tissue Cells Feel and Respond to the Stiffness of their Substrate. *Science* 310, 1139-1143 (2005). - Kumar, S. & Weaver, V. M. Mechanics, malignancy, and metastasis: The force journey of a tumor cell. *Cancer Metastasis Rev*. 28, 113-127 (2009). - Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. *Nature Reviews Molecular Cell Biology* 7, 265-275 (2006). - Bershadsky, A. D., Balaban, N. Q. & Geiger, B. Adhesion-dependent cell mechanosensitivity. *Annu. Rev. Cell Dev. Biol*. 19, 677-695 (2003). - Lecuit, T. & Lenne, P. Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesis. *Nature Reviews Molecular Cell Biology* 8, 633-644 (2007). - Johnson, C. P., Tang, H., Carag, C., Speicher, D. W. & Discher, D. E. Forced unfolding of proteins within cells. *Science* 317, 663-666 (2007). - Del Rio, A. et al. Stretching Single Talin Rod Molecules Activates Vinculin Binding. *Science* 323, 638-641 (2009). - Friedland, J. C., Lee, M. H. & Boettiger, D. Mechanically Activated Integrin Switch Controls alpha(5)beta(1) Function. *Science* 323, 642-644 (2009). - Na, S. et al. Rapid signal transduction in living cells is a unique feature of mechanotransduction. *Proc. Natl. Acad. Sci. U. S. A*. 105, 6626-6631 (2008). - Poh, Y. et al. Rapid Activation of Rac GTPase in Living Cells by Force Is Independent of Src RID C-9130-2011. *PLoS One* 4, e7886 (2009). - Dalby, M. J. et al. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. *Nature Materials* 6, 997-1003 (2007). - Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. *Cell* 126, 677-689 (2006). - Chowdhury, F. et al. Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells. *Nat. Mater*. 9, 82-8 (2010). - Na, S. & Wang, N. Application of Fluorescence Resonance Energy Transfer and Magnetic Twisting Cytometry to Quantify Mechanochemical Signaling Activities in a Living Cell. *Science Signaling* 1, pl1 (2008). - Poh YC et al. Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells. *Nature Commun*. In press. - Gall, J. G. CAJAL BODIES: The First 100 Years. *Annual Review of Cell & Developmental Biology* 16, 273 (2000). - Sleeman, J. & Lamond, A. Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway. *Current Biology* 9, 1065-1074 (1999). - Dundr, M. et al. In vivo kinetics of Cajal body components. *J. Cell Biol*. 164, 831-842 (2004). - Kaiser, T. E., Intine, R. V. & Dundr, M. De Novo Formation of a Subnuclear Body. *Science* 322, 1713-1717 (2008). - Platani, M., Goldberg, I., Lamond, A. I. & Swedlow, J. R. Cajal Body dynamics and association with chromatin are ATP-dependent. *Nat. Cell Biol*. 4, 502 (2002). - Wang, N., Butler, J. P. & Ingber, D. E. Mechanotransduction across the cell surface and through the cytoskeleton. *Science* 260, 1124 (1993). - Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. *Nature Methods* 3, 891-893 (2006). - Engler, A. et al. Substrate compliance versus ligand density in cell on gel responses. *Biophys. J*. 86, 617-628 (2004). - Yeung, T. et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. *Cell Motil. Cytoskeleton* 60, 24-34 (2005). - Tse, J. R. & Engler, A. J. Preparation of hydrogel substrates with tunable mechanical properties. *Current protocols in cell biology* / editorial board, Juan S.Bonifacino …[et al.] Chapter 10 (2010). - Tolic-Norrelykke, I. M., Butler, J. P., Chen, J. X. & Wang, N. Spatial and temporal traction response in human airway smooth muscle cells. *American Journal of Physiology-Cell Physiology* 283, C1254-C1266 (2002). - Butler, J. P., Tolic-Norrelykke, I. M., Fabry, B. & Fredberg, J. J. Traction fields, moments, and strain energy that cells exert on their surroundings. *American Journal of Physiology-Cell Physiology* 282, C595-C605 (2002). - Mijailovich, S. M., Kojic, M., Zivkovic, M., Fabry, B. & Fredberg, J. J. A finite element model of cell deformation during magnetic bead twisting. *J. Appl. Physiol*. 93, 1429-1436 (2002). - Fabry, B. et al. Signal transduction in smooth muscle – Selected contribution: Time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells. *J. Appl. Physiol*. 91, 986-994 (2001). - Truong, K. & Ikura, M. The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo. *Curr. Opin. Struct. Biol*. 11, 573-578 (2001). ### Acknowledgements The authors thank Dr. M. Dundr for help and discussion. The work was supported by NIH grant R01 GM072744. ### Associated Publications **Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells**. Yeh-Chuin Poh, Sergey P. Shevtsov, Farhan Chowdhury, Douglas C. Wu, Sungsoo Na, Miroslav Dundr, and Ning Wang. *Nature Communications* 3 () 29/05/2012 [doi:10.1038/ncomms1873](http://dx.doi.org/10.1038/ncomms1873) ### Author information **Yeh-Chuin Poh & Ning Wang**, University of Illnois Correspondence to: Ning Wang (nwangrw@illinois.edu) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2357) (2012) doi:10.1038/protex.2012.012. Originally published online 6 June 2012*.


 
 
 Find your black holes and white walls, know them … it is the only way you will be able to dismantle them and draw your lines of flight.—Deleuze and Guattari, A Thousand Plateaus
 
 
 Defined by originator David Belle as “an art to help you pass any obstacle”, the practice of “parkour” or “free running” constitutes both a mode of movement and a new way of interacting with the urban environment. Parkour was created by Belle (partly in collaboration with his childhood friend Sébastien Foucan) in France in the late 1980s. As seen in the following short video “Rush Hour”, a trailer for BBC One featuring Belle, parkour practitioners (known as “traceurs”), leap, spring, and vault from objects in the urban milieu that are intended to limit movement (walls, curbs, railings, fences) or that unintentionally hamper passage (lampposts, street signs, benches) through the space. 
 
 
 
 “Rush Hour” was among the first media representations of parkour, and it had a significant role in introducing and popularizing the practice in Britain. Parkour has subsequently been widely disseminated via news reports, Nike and Toyota ads, the documentaries Jump London (2003) and Jump Britain (2005), and feature films, including Luc Besson’s Yamakasi – Les Samouraïs des Temps Modernes (2001) and Banlieu 13 (2004; just released in the U.S. as District B13), starring David Belle as Leto and Cyril Raffaelli as Damien. Sébastien Foucan will appear in the upcoming James Bond film Casino Royale as Mollaka, a terrorist who is chased (parkour-style) and then killed by Bond. (Foucan can also be seen in the film’s trailer, currently available at both SonyPictures.com and AOL.com; the film itself is scheduled for release in November 2006). Madonna’s current “Confessions” tour features an extended parkour sequence (accompanying the song “Jump”), albeit one limited to the confines of a scaffold erected over the stage. Perhaps most important in the rapid development of parkour into a world-wide youth movement, however, has been the proliferation of parkour websites featuring amateur videos, photos, tutorials, and blogs. 
 
 The word “parkour” is derived from the French “parcours” (as the sport is known in France): a line, course, circuit, road, way or route, and the verb “parcourir”: to travel through, to run over or through, to traverse. As a physical discipline, parkour might be said to have a “poetics” — first, in general, in the Aristotelian sense of constructing through its various techniques (tekhnē) the drama of each parkour event. Secondly, one can consider parkour following Aristotle’s model of four-cause analysis as regards its specific materials (the body and the city), form or “vocabulary” of movements (drawn primarily from gymnastics, the martial arts, and modern dance), genre (as against, say, gymnastics), and purpose, including its effects upon its audience and the traceurs themselves. The existing literature on parkour (at this point, mostly news reports or websites) tends to emphasize the elements of form or movement, such as parkour’s various climbs, leaps, vaults, and drops, and the question of genre, particularly the ongoing, heated disputes among traceurs as to what is or is not true parkour. By contrast, my argument in this essay will focus principally on the materials and purpose of parkour: on the nature of the city and the body as they relate to parkour, and on the ways in which parkour can be seen to “remap” urban space and to demonstrate a resistance to its disciplinary functions, particularly as manifest in the urban street “grid.” 
 
 The institution of the street “grid” (or variations upon it such as Haussmann’s Parisian star-configuration) facilitates both the intelligibility — in terms of both navigation and surveillance — and control of space in the city. It situates people in urban spaces in determinate ways and channels the flow of pedestrian and vehicular traffic. The “grid” thus carries a number of normalizing and disciplinary functions, creating in effect what the philosophers Gilles Deleuze and Félix Guattari refer to as a “striation” of urban space. This striation constitutes “a process of capture of flows of all kinds, populations, commodities or commerce, money or capital, etc.” within a field of determinate spatial coordinates (Deleuze and Guattari 386). It establishes “fixed paths in well-defined directions, which restrict speed, regulate circulation, relativize movement, and measure in detail the relative movements of subjects and objects” (Deleuze and Guattari 386). Many of these aspects of striation can be seen in the ways urban space is depicted in the “Rush Hour” video: in the gridlocked traffic, the flashing tail-lights, the “STOP” light and “WAIT” sign, the sign indicating the proper directional flow of traffic, and the grim, bundled-up pedestrians trudging home en masse along the congested streets. 
 
 Against these images of conformity, regulation, and confinement, the video presents the parkour ethos of originality, “reach,” escape, and freedom. Belle’s (shirtless) aerial traversal of the urban space between his office and his flat — a swift, improvisational flow across the open rooftops (and the voids between them), off walls, and finally down the sloping roof into his apartment window — cuts across the striated space of the streets below and positions him, for that time, beyond the constrictions of the social realm and its “concrete” manifestations. Though parkour necessarily involves obstacles that must be “overcome,” the goal of parkour is to do this as smoothly and efficiently as possible, or, in the language of its practitioners, for the movement to be “fluid like water.” The experience of parkour might, then, be said to transform the urban landscape into “smooth space,” in Deleuze and Guattari’s sense of “a field without conduits or channels” (371), and thus into a space of uninhibited movement, at least in certain ideal moments. 
 
 Parkour seems to trace a path of desire (even if the desire is simply to avoid the crowds and get home in time to watch BBC One) that moves along a Deleuzean “line of flight,” a potential avenue of escape from the forces of striation and repression. Here the body is propelled over or through (most parkour movement actually takes place at ground level) the strata of urban space, perhaps with the hope that, as Deleuze and Guattari suggest, “one will bolster oneself directly on a line of flight enabling one to blow apart strata, cut roots, and make new connections” (15). In the process, parkour becomes “an art of displacement,” appropriating urban space in ways that temporarily disrupt their controlling logics and even imply the possibility of a smooth space of desire. 
 
 One might see parkour as an overcoming of social space (and its various constrictions and inhibitions of desire, its “stop” and “wait” signs) through the interplay of body and material barriers. The body becomes an instrument of freedom. This, again, is graphically conveyed in “Rush Hour” through the opening scene in which Belle strips off his business suit and through the subsequent repeated contrasts of his limber, revealed body to the rigid, swathed figures of the pedestrians below. In part an effect of the various camera angles from which it is shot, there is also an element of the “heroic” in this depiction of the body. This aspect of the representation appears to be knowingly acknowledged in the video’s opening sequence. The first frame is a close-up, tightly focused on a model of a ninja-like figure with a Japanese sword who first appears to be contemplating a building (with an out-of-focus Belle in the background contemplating it from the opposite direction), but then, in the next, full shot, is revealed to be scaling it — in the manner of superheroes and King Kong. The model remains in the frame as Belle undresses (inevitably evoking images of Clark Kent stripping down to his Superman costume) and, in the final shot of that sequence, the figure mirrors Belle’s as he climbs through the window and ascends the building wall outside. In the next sequence, Belle executes a breath-taking handstand on a guard railing on the edge of the roof with the panorama of the city behind him, his upper body spanning the space from the street to the edge of the city skyline, his lower body set against the darkening sky. 
 
 Through the practice of parkour, the relation between body and space is made dynamic, two reality principles in concert, interacting amid a suspension of the social strata. One might even say that the urban space is re-embodied — its rigid strata effectively “liquified.” In Jump London, the traceur Jerome Ben Aoues speaks of a Zen-like “harmony between you and the obstacle,” an idealization of what is sometimes described as a state of “flow,” a seemingly effortless immersion in an activity with a concomitant loss of self-consciousness. It suggests a different way of knowing the city, a knowledge of experience as opposed to abstract knowledge: parkour is, Jaclyn Law argues, “about curiosity and seeing possibilities — looking at a lamppost or bus shelter as an extension of the sidewalk” (np.). “You just have to look,” Sébastien Foucan insists in Jump London, “you just have to think like children….” Parkour effectively remaps urban space, creating a parallel, “ludic” city, a city of movement and free play within and against the city of obstacles and inhibitions. It reminds us that, in the words of the philosopher of urban space Henri Lefebvre, “the space of play has coexisted and still coexists with spaces of exchange and circulation, political space and cultural space” (172). Parkour tells us that in order to enter this space of play, we only need to make the leap.
 
 References
 
 Deleuze, Gilles and Félix Guattari. A Thousand Plateaus: Capitalism and Schizophrenia. Trans. Brian Massumi. Minneapolis: University of Minnesota Press, 1987. Jump London (2003). Mike Christie, director. Mike Smith, producer. Featuring Jerome Ben Aoues, Sébastien Foucan, and Johann Vigroux. Law, Jaclyn. “PK and Fly.” This Magazine May/June 2005 http://www.thismagazine.ca/issues/2005/05/>. Lefebvre, Henri. “Perspective or Prospective?” Writings on Cities. Trans. Eleonore Kofman and Elizabeth Lebas. Oxford: Blackwell, 1996. Rush Hour (2002). BBC One promotion trailer. Tom Carty, dir. Edel Erickson, pro. Produced by BBC Broadcast.
 See also:
 
 Wikipedia on parkour: http://www.en.wikipedia.org/wiki/Parkour> Parkour Worldwide Association: http://www.pawa.fr/> Parkour Net (multilingual): http://parkour.net/> NYParkour: http://www.nyparkour.com/> PKLondon.com: http://www.pklondon.com/> Nike’s “The Angry Chicken” (featuring Sébastien Foucan): http://video.google.com/videoplay? docid=-6571575392378784144&q=nike+chicken>
 There is an extensive collection of parkour videos available at YouTube 
 A rehearsal clip featuring Sébastien Foucan coaching the dancers for Madonna’s Confessions tour can be seen at YouTube 
 
 
 
 
 Citation reference for this article
 
 MLA Style
 Geyh, Paula. "Urban Free Flow: A Poetics of Parkour." M/C Journal 9.3 (2006). echo date('d M. Y'); ?> <http://journal.media-culture.org.au/0607/06-geyh.php>. APA Style
 Geyh, P. (Jul. 2006) "Urban Free Flow: A Poetics of Parkour," M/C Journal, 9(3). Retrieved echo date('d M. Y'); ?> from <http://journal.media-culture.org.au/0607/06-geyh.php>. 

Introduction For the past ten years, a transformative trend has emerged in the consumption of journalistic content, diverging significantly from its traditional engagement pathways. This evolution is characterised by the allure of serial journalistic podcasts such as Serial, which have seamlessly integrated narrative techniques typically reserved for fiction into journalistic storytelling (Kulkarni et al.). These podcasts have leveraged episodic structures, suspenseful build-ups, and dramatic climaxes to foster a level of engagement akin to fiction's grip on audiences. This shift towards addictive media consumption is eloquently linked to the binge-watching culture of on-demand television described by Dowling and Miller, situating such podcasts within a lineage of high-calibre television narratives (168). The concept of "binge-listening" (Krause and Uhrig 446) has emerged from this trend, signifying a consumption pattern where audiences, particularly those drawn to multi-part serial podcasts in genres like true crime, engage in extended listening sessions or eagerly anticipate new episodes. This pattern, reflective of an excessive indulgence in content, underscores the creators' success in crafting compelling narratives that captivate and retain audience interest. Illustrative of this phenomenon are listener testimonials for Die Mafiaprinzessin, a narrative podcast series by Süddeutsche Zeitung Magazine (part of Germany’s major quality newspaper), where users expressed their inability to disengage, consuming the entire series rapidly. One user stated, "I couldn't stop listening", and another, "I binged all episodes in two days" (quoted in Krause and Uhrig 447). But what makes serial storytelling podcasts in Germany so appealing, and which lessons can be drawn from this? This article provides answers through a historical lens and a focus on the creative process, distribution, and the transparency of journalistic research. Incorporating these sources and viewpoints, the discussion provides an overview of the cultural shift in media consumption towards serialised journalistic podcasts, highlighting their role in redefining audience engagement with media content. From Written to Spoken Storytelling Traditions The narrative-driven approach to audio journalism discussed in this article is typically unfolding over five to ten serialised podcast episodes of 30 to 45 minutes each, and diverges from shorter news snippets or dialogue-based formats, tackling complex topics through well-researched and dramatised storytelling. Kelleter defines serial storytelling as the delivery of continuation stories featuring consistent characters, produced and narrated in a mass-appealing, schematic manner (18). This definition, originally pertaining to fiction, aptly describes the serialised journalistic content's approach, emphasising that while the storytelling techniques may mirror fiction, the content remains firmly anchored in journalistic rigour and quality. It is neither based on a true story nor loosely associated with fiction. The podcast series that are the focus of this article are journalism in audio form, dedicated to journalism’s core values. This article aims to shed light on the development, cultural significance, and economic implications of such podcasts in Germany, specifically those produced by publishing houses as part of their digital strategy to gain digital subscriptions and hence turn readers into paying customers. The economic potential of such journalistic storytelling podcasts can be quite significant, as Newman describes in the 2023 Digital News Report: “podcasting may not yet be a mass market medium, but its audience profile is extremely interesting to publishers and to advertisers” (48). Newman continues to describe that podcast audiences generally have higher incomes, are more educated, and, notably, skew towards a younger demographic, making them an attractive demographic for publishers. While it is true that podcasts have achieved mass-market appeal on a global scale, the particular narratives that resonate with individual listeners can be highly specialised and varied, mirroring their diverse interests. This phenomenon is analogous to the realm of print magazines, which as a medium cater to a broad readership. However, individual publications often cater to distinct niches, attracting readers who share a specific set of interests. However, individual publications often target distinct niches, appealing to readers who share a specific set of interests. Podcasts in general appeal to a wide range of ages as a versatile medium, suitable for listening during various activities such as travel to and from work, dog walking, gym sessions, or while engaging in routine household chores like tidying up. Their capacity to build meaningful relationships with the audience is just beginning to be analysed. It has been found that “podcasts can provide informational and social gratifications to listeners” (Tobin and Guadagno 2). First Steps towards Serial Storytelling Podcasts in Germany The surge in serialised storytelling podcasts started shortly after the first season of the NPR podcast Serial in the English-speaking world, and was dubbed the medium’s “Golden Age” (Berry 170). These intricately produced journalistic podcasts became a new avenue for traditional media companies to market their in-depth research beyond print and online articles. And in many ways, this makes a lot of sense: with in-depth investigative research being one of the core values (and yet one of the most time-consuming, sometimes frustrating, and often very expensive assets) of any editorial medium, it makes economic sense to use as many channels as possible to publish the results of this research. In terms of content diversity, podcasts occupy a niche that is similar to what investigative journalism books or documentaries once did as a premium journalistic product where complex stories or investigations can be told in full, without the length constraints of typical journalistic formats (Krause and Uhrig 449). Podcasts have been distributed since 2005, but it took almost a decade for them to break away from the time limitations of linear radio slots. In Germany, serialised podcast storytelling arrived a year after Serial, with the Rundfunk Berlin-Brandenburg series Wer hat Burak erschossen? (Who Shot Burak?) from 2015, which many consider to be one of the first German podcast series in this new narrative tradition (Preger 7). Since then, the range of podcast series has diversified rapidly in Germany, just like in the US, in terms of both topics and providers. Following the already successful American themes of crime and terrorism, there were soon investigative research stories about topics ranging from the rise and fall of the former German economic powerhouse (and later notoriously fraudulent) Wirecard by Süddeutsche Zeitung or the popular media scandal about the publication of the fake diaries of Adolf Hitler by Stern Magazine in the 1980s, which the publishing house turned into a successful podcast series in 2020. And from 2021 onwards, there was an increase in biographically centred podcasts that combine elements of portraiture with investigative or contemporary historical elements such as the 2022 series Who the F*** Is Alice by Süddeutsche Zeitung Magazine, elaborating on the controversial work of Germany’s most popular first-generation feminist Alice Schwarzer. Yet, one of the most successful German storytelling podcasts is the episodic tale about the tragic descent of former radio host Ken Jebsen, from beloved (yet edgy) media personality to controversial conspiracy theorist, which was turned into a stunning tale in the series Cui Bono: WTF Happened to Ken Jebsen in 2021 (Eins 37), hitting a nerve in German society in times of Covid and the subsequent rise of populist conspiracy movements. Other notable German storytelling podcasts about prominent figures in Germany include the 2023 Series SchwarzRotGold: Mesut Özil zu Gast bei Freunden, about football player Mesut Özil and his complicated and highly political life story as an immigrant in Germany (published on RTL+), or Wild Wild Web: The Kim Dotcom Story from 2021 about Kim Dotcom, the controversial entrepreneur and founder of the now defunct file-sharing operator Megaupload, by Bayerischer Rundfunk. Specifics of Successful German Storytelling Podcasts While audio journalism has traditionally been a domain of public and private broadcasting companies in Germany, there has been a shift towards podcast productions from a more diverse set of media outlets. Approximately 66% of daily and weekly papers in Germany are currently producing podcasts, with 29% offering at least three different podcast series (Eins 104). This is a trend that is not limited to the big national subscription newspapers but can also be observed in smaller regional and local publications. According to a study by Wild & Wild, at least one third of the 308 regional and local subscription newspapers examined have incorporated podcasts into their offerings. The content of these podcasts primarily focusses on society and social issues (25%) and sports and leisure activities (20%) (175). What makes these podcasts specifically German (in contrast to series from other countries) is hard to answer and would require further research. What can be said, however, is that Germany has a vibrant scene for audio journalism and German audiences are rather familiar with the form of long-form audio reporting through the country's relatively strong public broadcasting system, which has been publishing quite elaborate forms of audio journalism since the 1950s. Even though many statistics and audience engagement metrics remain confidential, it has been written that audience engagement has been very good (Wild and Wild). It is evident that serial podcasts rank among the most successful digital offerings of large national media companies such as Süddeutsche Zeitung, fetching significant digital subscriptions with series like Wer ist Joni? (Who is Joni?), which was selected as one of the best podcasts in 2023 by the German newspaper Die Tageszeitung and was described as follows: Who is Joni? is a podcast about trust on the internet. We've all heard about marriage swindlers or people who extort money online. But Joni's case is different. It's particularly compelling because Christiane Lutz narrates it so personally. She contrasts her research with her own thoughts and feelings. She feels naïve, paranoid, angry, relieved, and all of this is completely relatable to the listener. (Fromm) As described here, the role of the host as the storyteller is paramount. The host serves as a convivial guide, offering subjective but meticulously researched narratives, sometimes paired with a serious sidekick for contrast. A recent study in Journalism Practice suggests that even news journalism benefits from narrative elements (Nee and Santana). Another study highlights two factors that enthral listeners: intimacy and emotion (Lindgren), which are prevalent across all podcast genres, including the often-criticised "chat podcasts" where two hosts discuss daily matters. At least in Germany, they are predominantly male, yet the masculine dominance has been challenged and reflected upon in recent discourse (Attig). These podcasts, which often rank highly on the German podcast charts on platforms such as Spotify, are quite different from the serial storytelling podcasts that many publishing houses see as a new way to engage with journalistic content. Common Ingredients of Successful German Storytelling Podcasts According to Schlütz, several characteristics distinguish narrative journalism as specific to podcasts, among them subjectivity, personalisation, contextualisation, and transparency (10). Building upon these findings, this article looks at various attributes of successful German podcasts from the serial storytelling variety. The selection of these podcasts was driven by their demonstrated popularity, as evidenced by reviews in newspapers, radio shows, or newsletters, as well as their recognition in the form of nominations or receipt of prestigious awards such as the German Reporter Prize and the German Podcast Prize. Such honours imply that these podcasts distinguish themselves by features like captivating storytelling, perceptive journalism, inventive production methods, or other exceptional qualities that have earned the respect and admiration of both their industry peers and listeners. Nevertheless, it should be acknowledged that this curated group does not represent an exhaustive overview of Germany's storytelling podcast landscape. The evaluation of the chosen podcasts was based on an analysis of their auditory content and the media's reception of them, including interviews with and reviews of both the podcasts and their creators. From this investigation, three principal insights emerged: Strong host figures who, in many cases, not only guide through the story, but become part of the story themselves. In the Norddeutscher Runkfunk production Eschede – 25 Jahre danach from 2023 (Eschede – 25 years after) the reporter Miriam Arndts researches the tragic accident that occurred in Eschede, Lower Saxony, in 1998, where a high-speed train derailed and collided with a bridge, resulting in the loss of 101 lives. Among the bereaved was Arndts, who lost her mother in the disaster. Making this podcast highly personal, Arndts engages with survivors and relatives of the victims, intertwining their accounts with her personal story, in line with Lindgren’s findings that “the involvement of the journalist (or host) in the story transcends self-reflexive metacommentary on journalistic practice to focus on the journalist as a private person” (10). Suspense and drama are leading elements of many of the successful podcasts: in Frauke Liebs – die Suche nach dem Mörder (Frauke Liebs – the search for the murderer) from the Magazine Stern, host and journalist Dominik Stawski embarks on a mission to solve a crime that he has been following for a good part of his career – addressing the murderer directly over the course of the episodes. This series could only be realised because of the reporter’s deep involvement in the story and his contacts with many of the people involved in the case, including the family of the victim. This is a good example of how such a series can be created from investigations that have already been published, but can now use the advantages of the longer form of serial storytelling in audio. The understanding of topical events and news is deepened by serial podcasts. As has been mentioned by Planer and Godulla, news stories can also be the drivers of in-depth audio storytelling (105). In Germany this can be exemplified by the popular series Die Flut – Warum musste Johanna sterben? (The flood – why did Johanna have to die?) from 2022, whose team of reporters from Westdeutscher Rundfunk investigates the circumstances of the tragic events of the deadly flood of the river Ahr in Germany that lead to many deaths in the summer of 2021. While this event is clearly of journalistic relevance, such tragedies are typically covered only for a short period by traditional news media. This podcast, in contrast, puts a lot of time and effort into trying to understand the consequences of such a natural disaster for those directly affected by it. The producers of the podcast describe their experience like this: During production, tears were shed more than once. And then, it is precisely this directness of emotion that makes the cruelty of the events tangible. It glosses over nothing, hides nothing. It makes the questions of responsibility directed at politicians even more pressing. (Beisenherz) These aspects show that – among others – these elements are recurring themes of storytelling podcasts in Germany. Of course, there are other factors that determine success of podcasts – the production (Preger 233), the distribution (Krause & Uhrig 457), and the marketing (Eins 169) being the most obvious ones. Current Trends and Economic Potential As Eins has pointed out, many leading publishers in Germany, including Der Spiegel, Süddeutsche Zeitung, and Handelsblatt, have in recent years created digital team positions to manage podcast productions (104). Audio studios have been established within publishing houses. Some editorial teams turn to external service providers, such as Die Zeit working with Pool Artists in Berlin for their podcasts or Süddeutsche Zeitung seeking external expertise for dramaturgy or sound design. The decision to manage podcasts in-house or to hire external providers depends on the available budget, expertise, and the complexity of the project (Eins 105). As Eins has pointed out, simple two-microphone interviews can be self-recorded by amateurs familiar with recording equipment and basic audio editing. However, sophisticated audio features with intricate sound design require teamwork, which is often sought from outside companies. German Media houses increasingly collaborate with external production companies. As podcast market competition grows, distinctive dramaturgy and sound may become crucial, especially for major media brands – this makes collaborations more interesting. For example, Süddeutsche Zeitung has produced elaborate investigative audio features, often in partnership with streaming services. The series Im Schattenkloster (2023/24) about a destructive religious cult in rural Bavaria has been produced in collaboration with Audible, an Amazon company. These podcast series sometimes continue the narrative of previously published investigations and articles in audio form, such as Der Spiegel's Made in Germany – das Flughafenfiasko BER or Going to Ibiza by Süddeutsche Zeitung, or they may be released as exclusive digital content like Narcoland by Aachener Zeitung. The appeal of producing serialised podcasts in an era where digital performance and the attraction of many paying customers to buy digital subscriptions is very important could be attributed to their relatively long production cycle (Krause and Uhrig 456). Unlike topical formats tied to release dates, complex storytelling podcasts retain significance over time in what is known as the Long Tail, described by Anderson, which encapsulated the concept of (digital) products that do not rely on high sales volume from a few mainstream products but achieve sales through a variety of niche products. And the numbers point in this direction: the 2020 podcast series Der Mörder und meine Cousine (The Murderer and My Cousin) produced by Bayerischer Rundfunk registered over 1.2 million plays between June 2020 and January 2022. Of these, 534,000 plays were recorded in the first two months following its release. Over the next 18 months, it garnered an additional 677,973 listens. Even in January 2022, more than 18 months post-release, the monthly play count exceeded 10,000, according to data from the provider's portal Spotify for Podcasters and the internal analysis tool MeFo Charts used by Bayerischer Rundfunk (Krause and Uhrig 457) Conclusion In the landscape of modern journalism in Germany, the advent of serial storytelling through podcasts has carved out a novel niche for high-quality narrative forms. Their appeal lies significantly in their adaptability, which has seen their popularity skyrocket and has made them an important asset in the sales strategy of digital subscriptions for publishing houses (Eins 106). By leveraging the power of audio, these formats not only captivate new demographics but also play a crucial role in shaping journalistic identities, presenting narratives in ways that resonate with the changing consumption patterns of listeners. These narratives, designed for on-the-go consumption, echo the episodic consumption trends set by intricate television and streaming shows, seamlessly fitting into the listener's lifestyle. Journalistically, these podcast series offer a compelling twist on storytelling (Krause and Uhrig 459), often expanding beyond traditional broadcast journalism principles to explore deeper, more intricate narratives, marking an exciting evolution in the way stories are told and consumed. This article has pointed out three aspects that storytelling podcasts in Germany have in common: strong host figures, suspense and drama, and a relation to current news and events, building bridges between classic news reporting and storytelling techniques exemplified by this article. It remains to be seen how these trends evolve in the future: the trend towards audio is described by the current Reuters Institute Digital News Report (Newman et al. 28) as “help[ing to] build loyal relationships, and ... good at attracting younger audiences”. Nevertheless, and despite all positive opportunities, there are of course limiting factors, such as the relatively high costs and long production cycles of such series, which require further investigation. References Anderson, Chris. The Long Tail: Why the Future of Business Is Selling Less of More. Hyperion, 2008. Attig, Christiane. "Männlich, Mittelalt, Gebildet – oder? Eine Charakterisierung deutschsprachiger Podcaster:Innen". Kommunikation@gesellschaft 21.2 (2020). Beisenherz, C. "Die Flut—Warum musste Johanna sterben?" 19 June 2023. <https://www1.wdr.de/podcast/die-flut-100.html>. Berry, Richard. "A Golden Age of Podcasting? Evaluating Serial in the Context of Podcast Histories." Journal of Radio & Audio Media 22.2 (2015): 170-178. Dowling, D.O., and K.J. Miller. "Immersive Audio Storytelling: Podcasting and Serial Documentary in the Digital Publishing Industry." Journal of Radio & Audio Media 26.1 (2019): 167-184. Eins, P. Podcasts im Journalismus: Eine Einführung für die Praxis. Springer Fachmedien, 2022. Fromm, A. "Recherche-Podcast ‘Wer ist Joni?’: Das einsame Mädchen." Die Tageszeitung, 22 Jan. 2023. <https://taz.de/!5907468/>. Kelleter, F. Populäre Serialität: Eine Einführung. Transcript Verlag, 2012. Krause, T., and K. Uhrig. "Journalismus zum Bingen: Potenziale und Funktionen serieller Podcasts für das digitale Storytelling." In Podcasts, eds. V. Katzenberger, J. Keil, and M. Wild. Wiesbaden: Springer Fachmedien, 2022. 445-460. Kulkarni, S., et al. "Innovating Online Journalism: New Ways of Storytelling." Journalism Practice 17.9 (2023): 845-1863. Lindgren, M. "Intimacy and Emotions in Podcast Journalism: A Study of Award-Winning Australian and British Podcasts." Journalism Practice 17.4 (2023): 704-719. Nee, R.C., and A.D. Santana. "Podcasting the Pandemic: Exploring Storytelling Formats and Shifting Journalistic Norms in News Podcasts Related to the Coronavirus." Journalism Practice 16.8 (2022) 1559-1577. Newman, N. "News Podcasts: Who Is Listening and What Formats Are Working?" In Reuters Institute Digital News Report 2023, eds. N. Newman et al. Oxford: Reuters Institute for the Study of Journalism, 2023. 48-52. Planer, R., and A. Godulla. "Storytelling in Podcasts deutscher Medienhäuser: Echte Interaktion, geplante Spontanität." In Podcasts, eds. V. Katzenberger, J. Keil, and M. Wild. Wiesbaden: Springer Fachmedien, 2022. 101-118. Preger, S. Geschichten erzählen: Storytelling für Radio und Podcast. Wiesbaden: Springer Fachmedien, 2019. Schlütz, D. "Auditive ‘Deep Dives’: Podcasts als narrativer Journalismus." Kommunikation@gesellschaft 21.2 (2020). Tobin, S.J., and R.E. Guadagno. "Why People Listen: Motivations and Outcomes of Podcast Listening." PLOS ONE 17.4 (2022). Wild, M., and T. Wild. "Vermessung der Podcastlandschaft: Eine explorative Analyse der Podcastangebote der Lokal- und Regionalzeitungen in Deutschland." In Podcasts: Perspektiven und Potenziale eines digitalen Mediums, eds. V. Katzenberger, J. Keil, and M. Wild. Wiesbaden: Springer Fachmedien, 2022. 153-179.