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Tiwari, Kiran B., Craig Gatto, Suzanne Walker, and Brian J. Wilkinson. "Exposure ofStaphylococcus aureusto Targocil Blocks Translocation of the Major Autolysin Atl across the Membrane, Resulting in a Significant Decrease in Autolysis." Antimicrobial Agents and Chemotherapy 62, no. 7 (2018): e00323-18. http://dx.doi.org/10.1128/aac.00323-18.
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ABSTRACTPeptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptibleStaphylococcus aureusstrain led to a rapid shut down of cellular autolysis. Targocil II, which targets the TarH subunit of TarGH, also resulted in a drastic decrease in autolysis. Here, we address the mechanism of targocil-mediated decreased autolysis. The mechanism is WTA dependent since targocil treatment decreased autolysis in methicillin-resistant strains but not in a WTA-deficient mutant. Similar to cellular autolysis, autolysin-retaining crude cell walls isolated from targocil-treated cells had vastly decreased autolytic activity compared to those from untreated cells. Purified cell walls from control and targocil-treated cells, which lack autolytic activity, were similarly susceptible to lysozyme and lysostaphin and had similar O-acetyl contents, indicating that targocil treatment did not grossly alter PG structure and chemistry. Purified cell walls from targocil-treated cells were highly susceptible to autolysin extracts, supporting the notion that targocil treatment led to decreased autolysin in the crude cell walls. Quantitative real-time PCR analysis revealed that the decrease in autolysis in the targocil-exposed cells was not due to transcriptional repression of the autolysin genesatl,lytM,lytN, andsle1. Zymographic analysis of peptidoglycan hydrolase profiles showed a deficiency of cell surface autolysins in targocil-treated cells but higher activity in cell membrane fractions. Here, we propose that the untranslocated WTA molecules in the targocil-exposed cells sequester Atl at the membrane, resulting in significantly decreased autolysis.
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Debeer, Sabine O. S., Thierry G. M. Baron, and Anna A. Bencsik. "Immunohistochemistry of PrPsc Within Bovine Spongiform Encephalopathy Brain Samples with Graded Autolysis." Journal of Histochemistry & Cytochemistry 49, no. 12 (2001): 1519–24. http://dx.doi.org/10.1177/002215540104901205.
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Bovine spongiform encephalopathy (BSE) is a transmissible neurodegenerative disease of cattle. Clinical diagnosis can be confirmed by investigation of both spongiform changes and abnormal prion protein (PrPsc), a marker considered specific for the disease. Tissue autolysis, often unavoidable in routine field cases, is not compatible with histological examination of the brain even though PrPsc is still detectable by immunoblotting. To determine how autolysis might affect accurate diagnosis using PrPsc immunohistochemistry, we studied 50 field samples of BSE brainstem (obex) with various degrees of autolysis. We demonstrated that the antigen-unmasking pretreatments necessary for PrPsc immunohistochemistry were compatible with the preservation of autolyzed brain sections and that PrPsc detection was unaffected by autolysis, even though anatomic markers were sometimes lost. In tissue samples in which anatomic sites were still recognizable, PrPsc accumulation was detected in specific gray matter nuclei. In samples with advanced autolysis, PrPsc deposits were still observed, at least at the cellular level, as an intraneuronal pattern. We found that the sensitivity of PrPsc immunohistochemistry as a diagnostic method for BSE was undiminished even by severe tissue autolysis.
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Shin, Kwang-Soo, Nak-Jung Kwon, Young Hwan Kim, Hee-Soo Park, Gi-Seok Kwon, and Jae-Hyuk Yu. "Differential Roles of the ChiB Chitinase in Autolysis and Cell Death of Aspergillus nidulans." Eukaryotic Cell 8, no. 5 (2009): 738–46. http://dx.doi.org/10.1128/ec.00368-08.
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ABSTRACT Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (ΔflbA) mutant autolyzed completely after a 3-day incubation, the ΔflbA ΔchiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the ΔflbA-induced autolysis. However, importantly, both ΔflbA and ΔflbA ΔchiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the ΔflbA and ΔflbA ΔchiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, β-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.
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Fournier, Bénédicte, and David C. Hooper. "A New Two-Component Regulatory System Involved in Adhesion, Autolysis, and Extracellular Proteolytic Activity ofStaphylococcus aureus." Journal of Bacteriology 182, no. 14 (2000): 3955–64. http://dx.doi.org/10.1128/jb.182.14.3955-3964.2000.
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ABSTRACT A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5′ terminus of the genearlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlSforms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, thearlS mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locusarlR-arlS strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence ofS. aureus.
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Escamez, Sacha, and Hannele Tuominen. "Contribution of cellular autolysis to tissular functions during plant development." Current Opinion in Plant Biology 35 (February 2017): 124–30. http://dx.doi.org/10.1016/j.pbi.2016.11.017.
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Guillen, F., F. Reyes, J. Rodriguez, and C. Vazquez. "Induction of an extracellular cellulase system during autolysis of Alternaria alternata." Transactions of the British Mycological Society 89, no. 1 (1987): 35–39. http://dx.doi.org/10.1016/s0007-1536(87)80054-2.
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Davis, Bill D. "ASSOCIATION OF α-AMYLASE WITH CELLULAR AUTOLYSIS IN PEA STEM TISSUES". American Journal of Botany 72, № 12 (1985): 1902–7. http://dx.doi.org/10.1002/j.1537-2197.1985.tb08463.x.
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Rouslin, W. "Persistence of mitochondrial competence during myocardial autolysis." American Journal of Physiology-Heart and Circulatory Physiology 252, no. 5 (1987): H985—H989. http://dx.doi.org/10.1152/ajpheart.1987.252.5.h985.
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The rate of irreversible loss of mitochondrial phosphorylating respiratory function with NAD-linked substrates during zero flow myocardial autolysis at 37 degrees C was gradual and relatively linear with time, progressing at about 1% of the control activity per minute. State 3 respiratory rates and initial rates of inner membrane potential development dropped off in close parallel with one another as well as with NADH-coenzyme Q (CoQ) reductase activity, suggesting that oxygen uptake as well as membrane potential development were rate limited by the increasing impairment of electron flow through complex I. Although the initial rate of membrane potential development dropped off gradually, the time course for the loss of the ability to ultimately develop and hold a full potential was slower still, there being only a moderate impairment of this ability at 80 min of autolysis. This sustained ability to develop and hold a membrane potential after more than 1 h of autolysis suggested that inner membrane leakiness contributed little or not at all to the functional impairment observed. The irreversible loss of mitochondrial inner membrane competence emerged in these studies as a relatively late development in the sequence of cellular alterations which characterize the myocardial ischemic process.
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Neumar, Robert W., Scott M. Hagle, Donald J. DeGracia, Gary S. Krause та Blaine C. White. "Brain μ-Calpain Autolysis During Global Cerebral Ischemia". Journal of Neurochemistry 66, № 1 (2002): 421–24. http://dx.doi.org/10.1046/j.1471-4159.1996.66010421.x.
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Roy, Rakesh, Ren-In You, Chan-Hua Chang, Chiou-Ying Yang, and Nien-Tsung Lin. "Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii." Microorganisms 9, no. 6 (2021): 1336. http://dx.doi.org/10.3390/microorganisms9061336.
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Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation–driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.
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Remacle, Albert G., Alexei V. Chekanov, Vladislav S. Golubkov, Alexei Y. Savinov, Dmitri V. Rozanov, and Alex Y. Strongin. "O-Glycosylation Regulates Autolysis of Cellular Membrane Type-1 Matrix Metalloproteinase (MT1-MMP)." Journal of Biological Chemistry 281, no. 25 (2006): 16897–905. http://dx.doi.org/10.1074/jbc.m600295200.
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Balentine, J. D., and W. B. Greene. "VESICULATION OF MYELIN IN POSTMORTEM AUTOLYSIS." Journal of Neuropathology and Experimental Neurology 45, no. 3 (1986): 363. http://dx.doi.org/10.1097/00005072-198605000-00148.
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Adam, Michael, Bao-Ling Tsay Adam, and Lloyd Wolfinbarger. "Ultrastructural characterization of the effects of beta-alanyl-melphalan in mouse Ehrlich ascites tumor cells and mouse liver cells." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (1992): 642–43. http://dx.doi.org/10.1017/s0424820100123611.
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Beta-alanyl-melphalan (BAM) was demonstrated to have significant cancerocidal activity using both in vitro cultures and in vivo chemotherapy assays. Ultrastructural observations in the present study confirmed the antitumor activity of melphalan (MEL) and BAM. Results showed that the MEL affected the cellular elements causing marked cell damage both in mouse Ehrlich ascites tumor cells (MEATC) and mouse liver cells (MLC). BAM appeared to affect the cellular elements, with marked cell damage, only in MEATC, but not in the MLC. There were nuclear and cytoplasmic alterations in MEL and BAM treated MEATC, i.e. peripheral thickening of chromatin in nuclei, severe mitochondrial alterations, ribosome accumulation and autolysis phenomena which lead to the destruction of the subcellular structures.
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Lu, Benjamin C. "Cell degeneration and gill remodelling during basidiocarp development in the fungus Coprinus cinereus." Canadian Journal of Botany 69, no. 6 (1991): 1161–69. http://dx.doi.org/10.1139/b91-149.
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The early mushroom gill development in a primordium of Coprinus cinereus was studied by electron microscopy. Extensive cell degeneration and cell death were found in gill cavities but not within gill domains. These degenerative cells were found to contain multivesicular and membranous residual bodies, suggesting that the multivesicular bodies are part of the cell degeneration. Cellular debris was observed in intercellular spaces probably as a consequence of cell lysis. The presence of multivesicular bodies was also observed in cells shortly before Coprinus basidiocarps underwent autolysis: a high dose of hydrolytic enzymes can be extracted from such basidiocarps. The high numbers of multivesicular bodies, the membranous residual bodies, and the cellular debris in the primordial tissues are manifestations of cell degeneration that may be a prerequisite to gill remodelling during early primordial development. Key words: cell degeneration, gill remodelling, multivesicular body, residual body, mushroom development.
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Tsuchido, T., T. Hiraoka, M. Takano, and I. Shibasaki. "Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids." Journal of Bacteriology 162, no. 1 (1985): 42–46. http://dx.doi.org/10.1128/jb.162.1.42-46.1985.
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Dhalluin, Anne, Ingrid Bourgeois, Martine Pestel-Caron, et al. "Acd, a peptidoglycan hydrolase of Clostridium difficile with N-acetylglucosaminidase activity." Microbiology 151, no. 7 (2005): 2343–51. http://dx.doi.org/10.1099/mic.0.27878-0.
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A gene encoding a putative peptidoglycan hydrolase was identified by sequence similarity searching in the Clostridium difficile 630 genome sequence, and the corresponding protein, named Acd (autolysin of C. difficile) was expressed in Escherichia coli. The deduced amino acid sequence of Acd shows a modular structure with two main domains: an N-terminal domain exhibiting repeated sequences and a C-terminal catalytic domain. The C-terminal domain exhibits sequence similarity with the glucosaminidase domains of Staphylococcus aureus Atl and Bacillus subtilis LytD autolysins. Purified recombinant Acd produced in E. coli was confirmed to be a cell-wall hydrolase with lytic activity on the peptidoglycan of several Gram-positive bacteria, including C. difficile. The hydrolytic specificity of Acd was studied by RP-HPLC analysis and MALDI-TOF MS using B. subtilis cell-wall extracts. Muropeptides generated by Acd hydrolysis demonstrated that Acd hydrolyses peptidoglycan bonds between N-acetylglucosamine and N-acetylmuramic acid, confirming that Acd is an N-acetylglucosaminidase. The transcription of the acd gene increased during vegetative cellular growth of C. difficile 630. The sequence of the acd gene appears highly conserved in C. difficile strains. Regarding deduced amino acid sequences, the C-terminal domain with enzymic function appears to be the most conserved of the two main domains. Acd is the first known autolysin involved in peptidoglycan hydrolysis of C. difficile.
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Monk, Ian R., Gregory M. Cook, Brian C. Monk, and Philip J. Bremer. "Morphotypic Conversion in Listeria monocytogenes Biofilm Formation: Biological Significance of Rough Colony Isolates." Applied and Environmental Microbiology 70, no. 11 (2004): 6686–94. http://dx.doi.org/10.1128/aem.70.11.6686-6694.2004.
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ABSTRACT Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.
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White, Stewart, David R. Berry, and Brian McNeil. "Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum." Journal of Biotechnology 75, no. 2-3 (1999): 173–85. http://dx.doi.org/10.1016/s0168-1656(99)00158-3.
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Kudzhaev, A. M., A. G. Andrianova, E. S. Dubovtseva, O. V. Serova та T. V. Rotanova. "Role of the Inserted α-Helical Domain in E. coli ATP-Dependent Lon Protease Function". Acta Naturae 9, № 2 (2017): 75–81. http://dx.doi.org/10.32607/20758251-2017-9-2-75-81.
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Multidomain ATP-dependent Lon protease of E. coli (Ec-Lon) is one of the key enzymes of the quality control system of the cellular proteome. A recombinant form of Ec-Lon with deletion of the inserted characteristic -helical HI(CC) domain (Lon-dHI(CC)) has been prepared and investigated to understand the role of this domain. A comparative study of the ATPase, proteolytic, and peptidase activities of the intact Lon protease and Lon-dHI(CC) has been carried out. The ability of the enzymes to undergo autolysis and their ability to bind DNA have been studied as well. It has been shown that the HI(CC) domain of Ec-Lon protease is required for the formation of a functionally active enzyme structure and for the implementation of protein-protein interactions.
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Ogata, J., C. Yutani, M. Imakita, et al. "Autolysis of the granular layer of the cerebellar cortex in brain death." Acta Neuropathologica 70, no. 1 (1986): 75–78. http://dx.doi.org/10.1007/bf00689517.
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Biz, Ana Paula, Elisandra Rigo, Angélica Patrícia Bertolo, and Darlene Cavalheiro. "Cellular disruption and its influence over the drying kinetics of brewer’s spent yeast biomass." Acta Scientiarum. Technology 42 (February 28, 2020): e48792. http://dx.doi.org/10.4025/actascitechnol.v42i1.48792.
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Spent yeast biomass is one of the residues of brewing. It is specifically the second-largest residue from brewing industry. Most of the spent yeast is sold at low prices, or disposed as waste or used as animal feed. Spent yeast biomass is predominantly composed of proteins, and it has a high biological value, being an excellent source of high-quality protein, comparable in value with soy protein. Therefore, spent yeast biomass has great potential for use in foodstuffs for human consumption. The objective of this work was to evaluate the influence of cell rupture over the drying kinetics of spent yeast biomass using mathematical models (Lewis and Page). Also, to verify the influence of cell rupture method over the amount of protein. The cellular rupture was performed by two methods (chemical method and physical method: ultrasound). The drying process was performed by freeze-drying, and the parameters of the models were obtained using the non-linear regression (Generalized Reduced Gradient Nonlinear Optimization Code). Mathematical models of drying kinetics showed a strong correlation with the experimental data, R² > 0.96. The disruption process did not significantly affect the drying time and protein content. But the cellular autolysis improves the protein digestibility since the proteins will be totally available to the digestive enzymes and also increase the bioavailability of nutrients.
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Udrisar, Daniel P., Maria I. Wanderley, Regina C. C. Porto, et al. "Androgen- and Estrogen-Dependent Regulation of Insulin-Degrading Enzyme in Subcellular Fractions of Rat Prostate and Uterus." Experimental Biology and Medicine 230, no. 7 (2005): 479–86. http://dx.doi.org/10.1177/153537020523000706.
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Innumerous data support the fact that insulin-degrading enzyme (IDE) is the primary enzymatic mechanism for initiating and controlling cellular insulin degradation. Nevertheless, insulin degradation is unlikely to be the only cellular function of IDE, because it appears that some cellular effects of insulin are mediated by IDE as a regulatory protein. Insulin-degrading enzyme shows a significant correlation with various cellular functions, such as cellular growth and differentiation, and the expression of IDE is developmentally regulated. Besides insulin, other substrates are also degraded by IDE, including various growth-promoting peptides. It has also been shown that IDE enhances the binding of androgen to DNA in the nuclear compartment. It is also known that the androgen hormones have a stimulatory effect on prostate growth, and that estradiol stimulates uterine growth. To establish whether IDE is regulated by a cellular prostate/uterine growth stimulus, the present study assessed whether IDE was modified in quantity and activity during proliferative conditions (castration + testosterone in the male rat, or castration + estradiol or the proestrus phase of the estrous cycle in the female rat) and autolysis (castration or the metestrus phase of the estrous cycle) using cytosolic and nuclear fractions of rat prostate and cytosolic fractions of rat uterus. The activity and amount of IDE decreased in the cytosolic fraction with castration and during metestrus, and increased with testosterone or estradiol treatment and during proestrus. In the nuclear fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although without degradative activity. The data presented here suggest that IDE may participate in prostatic and uterine growth and that the testosterone or estradiol and/or prostate and uterus insulin-like growth factors may be important factors for the expression and regulation of IDE in the prostate and uterus
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Ouyang, Jing, Fengjun Sun, Wei Feng, Yonghong Xie, Lijuan Ren, and Yongchuan Chen. "Antimicrobial Activity of Galangin and Its Effects on Murein Hydrolases of Vancomycin-Intermediate Staphylococcus aureus (VISA) Strain Mu50." Chemotherapy 63, no. 1 (2017): 20–28. http://dx.doi.org/10.1159/000481658.
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Backgroud: Antibiotic treatment for infections caused by vancomycin-intermediate Staphylococcus aureus (VISA) strains is challenging, and only a few effective and curative methods have been developed to combat these strains. This study aimed to investigate the antimicrobial activity of galangin against S. aureus and its effects on the murein hydrolases of VISA strain Mu50. This is the first report on these effects of galangin, and it may help to improve the treatment for VISA infections by demonstrating the effective use of galangin. Methods: Firstly, the minimum inhibitory concentration (MIC) and growth curve were used to investigate the antimicrobial activity of galangin against S. aureus. Secondly, transmission electron microscopy (TEM) was used to observe morphological changes of VISA strain Mu50. Thirdly, Triton X-100-induced autolysis and cell wall hydrolysis assays were performed to determine the activities of the murein hydrolases of Mu50. Finally, fluorescence real-time quantitative PCR was used to investigate the expression of the murein hydrolase-related Mu50 genes. Results: The results indicated that the MIC of galangin was 32 μg/mL against ATCC25293, N315, and Mu50, and galangin could significantly suppress the bacterial growth (p < 0.05) with concentrations of 4, 8 and 16 μg/mL, compared with control group (0 μg/mL). To explore the possible reasons of bacteriostatic effects of galangin, we observed morphological changes using TEM which showed that the division of Mu50 daughter cells treated with galangin was obviously inhibited. Considering the vital role of murein hydrolases in cellular division, assays were performed, and galangin markedly decreased Triton X-100-induced autolysis and cell wall hydrolysis. Galangin also significantly inhibited the expression of the murein hydrolase genes (atl, lytM, and lytN) and their regulatory genes (cidR, cidA, and cidB). Conclusions: Our findings indicated that galangin can effectively inhibit murein hydrolase activity as well as the growth of VISA strain Mu50.
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Tamber, Sandeep, Joseph Schwartzman, and Ambrose L. Cheung. "Role of PknB Kinase in Antibiotic Resistance and Virulence in Community-Acquired Methicillin-Resistant Staphylococcus aureus Strain USA300." Infection and Immunity 78, no. 8 (2010): 3637–46. http://dx.doi.org/10.1128/iai.00296-10.
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ABSTRACT The regulation of cellular processes by eukaryote-like serine/threonine kinases is widespread in bacteria. In the last 2 years, several studies have examined the role of serine/threonine kinases in Staphylococcus aureus on cell wall metabolism, autolysis, and virulence, mostly in S. aureus laboratory isolates in the 8325-4 lineage. In this study, we showed that the pknB gene (also called stk1) of methicillin-resistant S. aureus (MRSA) strain COL and the community-acquired MRSA (CA-MRSA) strain USA300 is involved in cell wall metabolism, with the pknB mutant exhibiting enhanced sensitivity to β-lactam antibiotics but not to other classes of antibiotics, including aminoglycosides, ciprofloxacin, bactrim, and other types of cell wall-active agents (e.g., vancomycin and bacitracin). Additionally, the pknB mutant of USA300 was found to be more resistant to Triton X-100-induced autolysis and also to lysis by lysostaphin. We also showed that pknB is a positive regulator of sigB activity, resulting in compromise in its response to heat and oxidative stresses. In association with reduced sigB activity, the expression levels of RNAII and RNAIII of agr and the downstream effector hla are upregulated while spa expression is downmodulated in the pknB mutant compared to the level in the parent. Consistent with an enhanced agr response in vitro, virulence studies of the pknB mutant of USA300 in a murine cutaneous model of infection showed that the mutant was more virulent than the parental strain. Collectively, our results have linked the pknB gene in CA-MRSA to antibiotic resistance, sigB activity, and virulence and have highlighted important differences in pknB phenotypes (virulence and sigB activity) between laboratory isolates and the prototypic CA-MRSA strain USA300.
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Hewitt, Kimberley E., Howard J. Lesiuk, Joseph S. Tauskela, Paul Morley, and Jon P. Durkin. "Selective coupling of ?-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons." Journal of Neuroscience Research 54, no. 2 (1998): 223–32. http://dx.doi.org/10.1002/(sici)1097-4547(19981015)54:2<223::aid-jnr10>3.0.co;2-5.
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McKersie, Bryan D., R. L. Peterson, Stephen R. Bowley, and Shankar Das. "Ultrastructural and genetic characterization of a mutant exhibiting starch accumulation and premature leaf senescence in Medicago sativa." Canadian Journal of Botany 70, no. 11 (1992): 2245–53. http://dx.doi.org/10.1139/b92-278.
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A mutant was isolated from irradiated seed of alfalfa, Medicago sativa L. cv. Excalibur. The mutant plant, Ex-139, displayed symptoms of premature senescence in the leaf palisade mesophyll. The leaves emerged as a normal phenotype, but light microscopy revealed that they rapidly began to accumulate starch in plastids of some cells in the palisade mesophyll. This accumulation of starch was followed by general cellular autolysis leading to the formation of necrotic regions in the palisade mesophyll. The adjacent epidermal and spongy mesophyll cells were not structurally affected. The mutant otherwise exhibited normal growth and development and was fertile. Inheritance studies indicated that the trait was transmitted to the progeny, preferentially but not exclusively, through the female, which suggests that either there is differential selection among male and female gametes or the trait is controlled by extranuclear DNA. This mutant should be useful in the study of the link between senescence and carbohydrate metabolism and in alfalfa genetics. Key words: starch metabolism, plastid, chloroplast genome, biparental inheritance.
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Wodzicki, Tomasz J. "Forest – the photosphere of life in the Earth’s atmosphere." Forest Research Papers 81, no. 3 (2020): 133–38. http://dx.doi.org/10.2478/frp-2020-0015.
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Abstract The evolution of the vertical, long distance water transport, overcoming gravitation, by trees during the Devonian, initiated the emerging of forest ecosystems extending the photosphere of life further into the Earth's atmosphere. The origin of woody tissues is likely associated with genome mutations in primitive green plants, which inhabited the land about 350 million years ago. Most probably, only two mutations were required – one allowing the synthesis of lignin and the second, enabling the autolysis of protoplast in the maturing cellular woody elements. Developing forest ecosystems formed the most productive environments, in which sunlight-dependent metabolic processes of life reached further into the atmosphere while at the same time allowing more water to be stored on the land surface, which in turn allowed for the evolution of numerous heterotrophic organisms. This property of the forest could therefore be considered an important factor in the evolution of hominids, which eventually contributed to the development of the Homo sapiens culture.
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Waters, Christopher M., Michelle H. Antiporta, Barbara E. Murray, and Gary M. Dunny. "Role of the Enterococcus faecalis GelE Protease in Determination of Cellular Chain Length, Supernatant Pheromone Levels, and Degradation of Fibrin and Misfolded Surface Proteins." Journal of Bacteriology 185, no. 12 (2003): 3613–23. http://dx.doi.org/10.1128/jb.185.12.3613-3623.2003.
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ABSTRACT Gelatinase (GelE), a secreted Zn-metalloprotease of Enterococcus faecalis, has been implicated as a virulence factor by both epidemiological data and animal model studies. Expression of gelE is induced at a high cell density by the fsr quorum-sensing system. In the present study, GelE was shown to be responsible for the instability of a number of Asc10 (aggregation substance) mutant proteins, implying that GelE functions to clear the bacterial cell surface of misfolded proteins. Disruption of GelE production led to increased cell chain length of E. faecalis, from a typical diplococcus morphology to chains of 5 to 10 cells. This function of GelE was also exhibited when the protein was expressed in Streptococcus pyogenes. GelE-expressing E. faecalis strains were more autolytic, suggesting that GelE affects chain length through activation of an autolysin. GelE was also essential for degradation of polymerized fibrin. GelE expression reduced the titer of cCF10, the peptide pheromone that induces conjugation of pCF10, and pCF10 had increased conjugation into non-GelE-expressing strains. These new functions attributed to GelE suggest that it acts to increase the dissemination of E. faecalis in high-density environments.
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Fang, Eric G. C., and Ralph A. Dean. "Site-Directed Mutagenesis of the magB Gene Affects Growth and Development in Magnaporthe grisea." Molecular Plant-Microbe Interactions® 13, no. 11 (2000): 1214–27. http://dx.doi.org/10.1094/mpmi.2000.13.11.1214.
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G protein signaling is commonly involved in regulating growth and differentiation of eukaryotic cells. We previously identified MAGB, encoding a Gα subunit, from Magnaporthe grisea, and disruption of MAGB led to defects in a number of cellular responses, including appres-sorium formation, conidiation, sexual development, mycelial growth, and surface sensing. In this study, site-directed mutagenesis was used to further dissect the pleiotropic effects controlled by MAGB. Conversion of glycine 42 to arginine was predicted to abolish GTPase activity, which in turn would constitutively activate G protein signaling in magBG42R. This dominant mutation caused autolysis of aged colonies, misscheduled melanization, reduction in both sexual and asexual reproduction, and reduced virulence. Furthermore, magBG42R mutants were able to produce appressoria on both hydrophobic and hydrophilic surfaces, although development on the hydrophilic surface was delayed. A second dominant mutation, magBG203R (glycine 203 converted to arginine), was expected to block dissociation of the Gβγ from the Gα subunit, thus producing a constitutively inactive G protein complex. This mutation did not cause drastic phenotypic changes in the wild-type genetic background, other than increased sensitivity to repression of conidiation by osmotic stress. However, magBG203R is able to complement phenotypic defects in magB mutants. Comparative analyses of the phenotypical effects of different magB mutations are consistent with the involvement of the Gβγ subunit in the signaling pathways regulating cellular development in M. grisea.
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SHIELDS, DONALD C., CHRISTINE LEBLANC, and NAREN L. BANIK. "Calcium-Mediated Neurofilament Protein Degradation in Rat Optic Nerve In Vitro: Activity and Autolysis of Calpain Proenzyme." Experimental Eye Research 65, no. 1 (1997): 15–21. http://dx.doi.org/10.1006/exer.1997.0286.
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Wolska, Krystyna, Anna Grudniak, Beata Fiecek, Anna Kraczkiewicz-Dowjat, and Anna Kurek. "Antibacterial activity of oleanolic and ursolic acids and their derivatives." Open Life Sciences 5, no. 5 (2010): 543–53. http://dx.doi.org/10.2478/s11535-010-0045-x.
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AbstractBacterial resistance to antibiotics is increasing at an alarming rate and many commonly used antibiotics are no longer effective. Thus, there is considerable interest in investigating novel antibacterial compounds, such as the plant-derived pentacyclic triterpenoids, including oleanolic acid (OA), ursolic acid (UA) and their derivatives. These compounds can be isolated from many medicinal and crop plants and their antibacterial, antiviral, antiulcer and anti-inflammatory effects are well documented. OA and UA are active against many bacterial species, particularly Gram-positive species, including mycobacteria. They inhibit bacterial growth and survival, and the spectrum of minimal inhibitory concentration (MIC) values is very broad. In addition, OA, UA and their derivatives display potent antimutagenic activity. Studies to identify the cellular targets and molecular mechanisms of OA and UA action were initiated a few years ago and it has already been demonstrated that both acids influence bacterial gene expression, the formation and maintenance of biofilms, cell autolysis and peptidoglycan turnover. Before these compounds can be used clinically as antimicrobial agents, further extensive studies are required to determine their cytotoxicity and the optimum mode of their application.
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Raafat, Dina, Kristine von Bargen, Albert Haas, and Hans-Georg Sahl. "Insights into the Mode of Action of Chitosan as an Antibacterial Compound." Applied and Environmental Microbiology 74, no. 12 (2008): 3764–73. http://dx.doi.org/10.1128/aem.00453-08.
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ABSTRACT Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.
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Amrolia, Persis J., Robert Wynn, Rachael Hough, et al. "Simultaneous Targeting of CD19 and CD22: Phase I Study of AUTO3, a Bicistronic Chimeric Antigen Receptor (CAR) T-Cell Therapy, in Pediatric Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL): Amelia Study." Blood 132, Supplement 1 (2018): 279. http://dx.doi.org/10.1182/blood-2018-99-118616.
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Abstract Introduction CAR T-cell therapies directed against CD19 or CD22 antigens have shown significant activity in pediatric patients with r/r B-ALL. Whilst complete response (CR) rates of 70‒90% have been observed, relapse due to target antigen downregulation or loss is the major cause of treatment failure. This Phase I/II study evaluates the safety and efficacy of AUTO3, a CAR T-cell therapy designed to target CD19 and CD22 simultaneously in order to reduce the likelihood of relapse due to antigen loss. Methods & Patients We constructed a novel bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. Antigen binding domains were humanized and both CARs are in "2nd generation" format (incorporating an OX40 co-stimulatory domain for the CD19 CAR and a 41BB for the CD22 CAR). The performance of the CD22 CAR was optimized by incorporating a novel pentameric spacer. The cell product was manufactured on a semi-automated and closed process using CliniMACS® Prodigy (Miltenyi Biotec). Patients (1‒24 years of age) with high risk relapse (IBFM criteria) or relapse post-allogeneic stem cell transplant (SCT), adequate performance score/organ function, and an absolute lymphocyte count ≥0.5 x 109/L are eligible. Patients with CNS 3 disease, active graft versus host disease, and clinically significant infection or serious toxicity from prior CAR T-cell are excluded. Patients receive lymphodepletion with 30 mg/m2/day fludarabine x 4 days and 500 mg/m2/day cyclophosphamide x 2 days prior to AUTO3 infusion. Three dose levels are being explored (1 x 106, 3 x 106, and 5 x 106 transduced CAR+ T cells/kg) and CAR T cells are infused as a single (for <25% blasts) or split (for >25% blasts) dose based on leukemia burden. Bridging therapy is allowed during the manufacturing period. The primary endpoint of Phase I is the frequency of dose-limiting toxicities (DLTs) and key secondary endpoints include proportion of patients achieving a morphological/minimal residual disease (MRD) negative CR, disease-free survival, overall survival, as well as biomarker endpoints including AUTO3 levels and persistence in blood and bone marrow. Results As of the data cut-off date (July 16, 2018), 9 patients have been enrolled and 8 have received AUTO3. It was possible to generate a product in all patients and the median transduction efficiency was 16% (range 9‒34%). Median age was 7.5 years (range 4‒16 years) and 5 (63%) patients had prior SCT. One patient (13%) had prior anti-CD19 CAR-T cells and blinatumomab. The disease burden at Day ‒7 ranged from 0% to 90% leukemic blasts. Eight patients had a minimum of 4 weeks' follow up and were evaluable for safety and efficacy analysis. Three patients received ≤1 × 106 CAR T cells/kg as single dose, 1 patient received 2 × 106/kg as split dose, and 4 received 3 × 106 CAR cells/kg (3 single infusions, 1 split). No AUTO3 related deaths and no DLTs were observed. The most common grade (Gr) ≥3 adverse events were neutropenia (63%), febrile neutropenia (50%), pyrexia (25%), and anemia (25%). Five patients (63%) had Gr 1 cytokine release syndrome (CRS); no Gr 2 or higher CRS was seen. Five patients (63%) experienced neurotoxicity: 4 had Gr 1 and 1 patient (13%) had Gr 3 encephalopathy that was considered likely related to prior intrathecal methotrexate. No patients required ICU admission. Six of 8 patients achieved MRD negative CR, giving an objective response rate of 75% (95% CI 34.9‒96.8%) at 1 month. In patients treated at doses <3 x 106/kg, 3 responded but subsequently relapsed. Importantly, no loss of CD19 or CD22 was noted in patients that relapsed. All 4 patients treated at the higher dose of 3 × 106 CAR T cells/kg had an MRD negative CR with ongoing remission and B-cell aplasia, with the longest follow up of 4 months. CAR T-cell expansion was enhanced in patients receiving 3 x 106/kg (median 79,282 copies/µg DNA in blood at peak) compared to those receiving lower doses (median 10,243 copies/µg DNA). Conclusion This interim data analysis demonstrates for the first time the feasibility and safety of simultaneous targeting of CD19 and CD22 with AUTO3. Promising efficacy was demonstrated at a dose level of 3 × 106 CAR T cells/kg, as 4/4 patients achieved MRD complete remission with no antigen negative escape at this early stage. The study continues to enrol and updated follow up and additional patient data at higher dose levels, as well as cellular kinetics and additional biomarker analysis, will be presented. Disclosures Wynn: Orchard SAB: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Equity Ownership; Chimerix: Research Funding; Genzyme: Honoraria; Bluebird Bio: Consultancy; Orchard Therapeutics: Consultancy; Chimerix: Consultancy. Hough:University College London Hospital's NHS Foundation Trust: Employment. Vora:Amgen: Other: Advisory board; Medac: Other: Advisory board; Novartis: Other: Advisory board; Pfizer: Other: Advisory board; Jazz: Other: Advisory board. Veys:Servier: Research Funding; Pfizer: Honoraria; Novartis: Honoraria. Chiesa:Gilead: Consultancy; Bluebird Bio: Consultancy. Al-Hajj:Autolus Ltd: Employment; Autolus Ltd: Equity Ownership. Cordoba:Autolus Ltd: Employment; Autolus Ltd: Equity Ownership; Autolus Ltd: Patents & Royalties. Onuoha:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Kotsopoulou:Autolus Ltd: Equity Ownership; Autolus Ltd: Employment. Khokhar:Autolus Ltd: Employment; Autolus Ltd: Equity Ownership. Pule:Autolus Ltd: Employment, Equity Ownership, Other: Salary contribution paid for by Autolus, Research Funding; University College London: Patents & Royalties: Patent with rights to Royalty share through UCL. Peddareddigari:Autolus Therapeutics plc: Equity Ownership; Autolus Therapeutics plc: Patents & Royalties; Autolus Therapeutics plc: Employment.
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Johnson, Brant R., та Todd R. Klaenhammer. "AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM". Applied and Environmental Microbiology 82, № 18 (2016): 5687–97. http://dx.doi.org/10.1128/aem.02025-16.
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ABSTRACTAutolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome ofLactobacillus acidophilusNCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins inL. acidophilusNCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the genelba0176(acmB), was selected for functional analysis.In silicoanalysis revealed thatacmBorthologs are found exclusively in S-layer- forming species ofLactobacillus. Chromosomal deletion ofacmBresulted in aberrant cell division, autolysis, and autoaggregation. Complementation ofacmBin the ΔacmBmutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmBstrain for mucin and extracellular matrices fibronectin, laminin, and collagenin vitro. These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface ofL. acidophilusNCFM and other S-layer-producingLactobacillusspecies.IMPORTANCELactobacillus acidophilusis one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.
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Burgess Tornaletti, Laura, and Giulia Manina. "Delving Into the Functional Meaning of Phenotypic Variation in Mycobacterial Persistence: Who Benefits the Most From Programmed Death of Individual Cells?" Microbiology Insights 13 (January 2020): 117863612094530. http://dx.doi.org/10.1177/1178636120945304.
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The lengthy tuberculosis therapy is emblematic of how hard drug-persistent infections are to eradicate. Phenotypic variation within clonal bacterial communities contributes to drug evasion and has major implications for the treatment of drug-persistent infections. We reported that single mycobacterial cells exhibit differential drug susceptibility, contingent on their inherent phenotypic variation in DNA damage response. Individual cells experiencing severe DNA damage massively induce the SOS response and exhibit signs of programmed cell death (PCD), such as unbalanced growth, chromosomal fragmentation, autolysis, and release of the intracellular content. Toxin-antitoxin systems are known to contribute to PCD in model microorganisms by targeting essential cellular processes, and they might function similarly in mycobacteria. We have found that the toxin MazF and a Clp protease, possibly responsible for degrading the MazF cognate antitoxin MazE, are induced during harsh conditions in a model organism for tuberculosis, and that cells that are about to lyse from drug exposure display a buildup of toxin. Deeper analysis of PCD in mycobacteria may reveal whether this process belongs to a broader strategy for the community’s survival. Finally, disrupting the balance between survival and PCD may prove useful to tackle drug evasion in mycobacterial persistent subpopulations.
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Shiraishi, T., S. Yokota, Y. Sato, et al. "Lipoteichoic acids are embedded in cell walls during logarithmic phase, but exposed on membrane vesicles in Lactobacillus gasseri JCM 1131T." Beneficial Microbes 9, no. 4 (2018): 653–62. http://dx.doi.org/10.3920/bm2017.0124.
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Lipoteichoic acid (LTA) is a cell surface molecule specific to Gram-positive bacteria. How LTA localises on the cell surface is a fundamental issue in view of recognition and immunomodulation in hosts. In the present study, we examined LTA localisation using strain JCM 1131T of Lactobacillus gasseri, which is a human intestinal lactic acid bacterium, during various growth phases by immunoelectron microscopy. We first evaluated the specificity of anti-LTA monoclonal antibody clone 55 used as a probe. The glycerophosphate backbone comprising almost intact size (20 to 30 repeating units) of LTA was required for binding. The antibody did not bind to other cellular components, including wall-teichoic acid. Immunoelectron microscopy indicated that LTA was embedded in the cell wall during the logarithmic phase, and was therefore not exposed on the cell surface. Similar results were observed for Lactobacillus fermentum ATCC 9338 and Lactobacillus rhamnosus ATCC 7469T. By contrast, membrane vesicles were observed in the logarithmic phase of L. gasseri with LTA exposed on their surface. In the stationary and death phases, LTA was exposed on cell wall-free cell membrane generated by autolysis. The dramatic alternation of localisation in different growth phases and exposure on the surface of membrane vesicles should relate with complicated interaction between bacteria and host.
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Bæk, Kristoffer T., Angelika Gründling, René G. Mogensen та ін. "β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus USA300 Is Increased by Inactivation of the ClpXP Protease". Antimicrobial Agents and Chemotherapy 58, № 8 (2014): 4593–603. http://dx.doi.org/10.1128/aac.02802-14.
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ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) has acquired themecAgene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for β-lactams. Quickly spreading and highly virulent community-acquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the β-lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating β-lactam resistance. These pathways do not involve the control ofmecAexpression, as the cellular levels of PBP2a were unaltered in theclpmutants. An analysis of the cell envelope properties of theclpXandclpPmutants revealed a number of distinct phenotypes that may contribute to the enhanced β-lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in theclpmutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent β-lactam resistance.
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Sieradzki, Krzysztof, Paolo Villari, and Alexander Tomasz. "Decreased Susceptibilities to Teicoplanin and Vancomycin among Coagulase-Negative Methicillin-Resistant Clinical Isolates of Staphylococci." Antimicrobial Agents and Chemotherapy 42, no. 1 (1998): 100–107. http://dx.doi.org/10.1128/aac.42.1.100.
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ABSTRACT Of 41 methicillin-resistant coagulase-negative staphylococcal clinical isolates collected during a 5-month period between late 1995 and early 1996, 28 showed tube dilution teicoplanin MICs of 4 to 8 μg/ml which increased to 16 to 32 μg/ml upon prolonged incubation. Cultures of such bacteria were heterogeneous; they contained subpopulations with frequencies of 10−5 to 10−4 that could grow on up to 50 μg of teicoplanin per ml. The same cultures were also heterogeneous with respect to susceptibility to vancomycin; while the MICs for the majority of cells were 2 to 4 μg/ml, subpopulations that could grow on 6 to 12 μg of vancomycin per ml were also present at frequencies of 10−5to 10−7. Selective enrichment of such cultures for the resistant subpopulation occurred with relative ease under laboratory conditions. Heterogeneous phenotypes for teicoplanin (but not for vancomycin) susceptibility were also identified in severalStaphylococcus epidermidis isolates collected during the preantibiotic era. The addition of half the MIC of teicoplanin inhibited autolysis and caused formation of cellular aggregates which disintegrated to individual bacteria in the stationary phase when the titer of teicoplanin in the medium fell to undetectable levels, indicating removal of the antibiotic from the culture medium by the bacteria.
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Yeh, Chu, Liu, and Chen. "Differential Gene Profiling of the Heartwood Formation Process in Taiwania cryptomerioides Hayata Xylem Tissues." International Journal of Molecular Sciences 21, no. 3 (2020): 960. http://dx.doi.org/10.3390/ijms21030960.
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Taiwania (Taiwania cryptomerioides) is an important tree species in Taiwan because of the excellent properties of its wood and fascinating color qualities of its heartwood (HW), as well as the bioactive compounds therein. However, limited information is available as to the HW formation of this species. The objective of this research is to analyze the differentially expressed genes (DEGs) during the HW formation process from specific Taiwania xylem tissues, and to obtain genes that might be closely associated with this process. The results indicated that our analyses have captured DEGs representative to the HW formation process of Taiwania. DEGs related to the terpenoid biosynthesis pathway were all up-regulated in the transition zone (TZ) to support the biosynthesis and accumulation of terpenoids. Many DEGs related to lignin biosynthesis, and two DEGs related to pinoresinol reductase (PrR)/pinoresinol lariciresinol reductase (PLR), were up-regulated in TZ. These DEGs together are likely involved in providing the precursors for the subsequent lignan biosynthesis. Several transcription factor-, nuclease-, and protease-encoding DEGs were also highly expressed in TZ, and these DEGs might be involved in the regulation of secondary metabolite biosynthesis and the autolysis of the cellular components of ray parenchyma cells in TZ. These results provide further insights into the process of HW formation in Taiwania.
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Cora, Michelle C., Kyathanahalli S. Janardhan, Heather Jensen, Natasha Clayton, and Gregory S. Travlos. "Previously Diagnosed Reticulum Cell Hyperplasia in Decalcified Rat Bone Marrow Stain Positive for Ionized Calcium Binding Adapter Molecule 1 (Iba1): A Monocytic/Macrophage Cell Marker." Toxicologic Pathology 48, no. 2 (2019): 317–22. http://dx.doi.org/10.1177/0192623319890610.
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Reticulum cell hyperplasia (RCH) was a term used for many years by the National Toxicology Program (NTP) to describe a certain non-neoplastic bone marrow lesion of rats. Retrospective microscopic evaluation of RCH lesions and immunohistochemistry analyses were performed to reassess and further characterize these lesions. The NTP database was searched to identify femoral bone marrow specimens diagnosed with RCH from 1981 to 2014 (n = 254). The diagnosis last occurred in 2003, after which the term “cellular infiltration” was used. Eighty-three RCH slides, spanning 22 years, representing 34 different chemicals, were selected for microscopic review, and a subset (23) was chosen for ionized calcium binding adapter molecule 1 (Iba1) immunohistochemical staining; initial investigations revealed Iba1 worked as a macrophage marker on decalcified tissue. The following diagnoses were made upon reevaluation: 36 were consistent with cellularity increased, macrophage, 22 with histiocytic sarcoma, 8 with increased myeloid cells, 4 with autolysis, and 13 were normal appearance. All 23 RCH lesions stained positive for Iba1. Fifty-eight of 83 bone marrows previously diagnosed with RCH are consistent morphologically and immunohistochemically with cells of histiocytic origin. These results will help with interpretation of historical data and demonstrates that Iba1 can be used in decalcified bone marrow sections.
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Andre, Guillaume, Kees Leenhouts, Pascal Hols, and Yves F. Dufrêne. "Detection and Localization of Single LysM-Peptidoglycan Interactions." Journal of Bacteriology 190, no. 21 (2008): 7079–86. http://dx.doi.org/10.1128/jb.00519-08.
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ABSTRACT The lysin motif (LysM) is a ubiquitous protein module that binds peptidoglycan and structurally related molecules. Here, we used single-molecule force spectroscopy (SMFS) to measure and localize individual LysM-peptidoglycan interactions on both model and cellular surfaces. LysM modules of the major autolysin AcmA of Lactococcus lactis were bound to gold-coated atomic force microscopy tips, while peptidoglycan was covalently attached onto model supports. Multiple force curves recorded between the LysM tips and peptidoglycan surfaces yielded a bimodal distribution of binding forces, presumably reflecting the occurrence of one and two LysM-peptidoglycan interactions, respectively. The specificity of the measured interaction was confirmed by performing blocking experiments with free peptidoglycan. Next, the LysM tips were used to map single LysM interactions on the surfaces of L. lactis cells. Strikingly, native cells showed very poor binding, suggesting that peptidoglycan was hindered by other cell wall constituents. Consistent with this notion, treatment of the cells with trichloroacetic acid, which removes peptidoglycan-associated polymers, resulted in substantial and homogeneous binding of the LysM tip. These results provide novel insight into the binding forces of bacterial LysMs and show that SMFS is a promising tool for studying the heterologous display of proteins or peptides on bacterial surfaces.
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Duque, Cristiane, Rafael N. Stipp, Bing Wang, et al. "Downregulation of GbpB, a Component of the VicRK Regulon, Affects Biofilm Formation and Cell Surface Characteristics ofStreptococcus mutans." Infection and Immunity 79, no. 2 (2010): 786–96. http://dx.doi.org/10.1128/iai.00725-10.
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ABSTRACTThe virulence of the dental caries pathogenStreptococcus mutansrelies in part on the sucrose-dependent synthesis of and interaction with glucan, a major component of the extracellular matrix of tooth biofilms. However, the mechanisms by which secreted and/or cell-associated glucan-binding proteins (Gbps) produced byS. mutansparticipate in biofilm growth remain to be elucidated. In this study, we further investigate GbpB, an essential immunodominant protein with similarity to murein hydrolases. A conditional knockdown mutant that expressedgbpBantisense RNA under the control of a tetracycline-inducible promoter was constructed in strain UA159 (UACA2) and used to investigate the effects of GbpB depletion on biofilm formation and cell surface-associated characteristics. Additionally, regulation ofgbpBby the two-component system VicRK was investigated, and phenotypic analysis of avicKmutant (UAvicK) was performed. GbpB was directly regulated by VicR, and several phenotypic changes were comparable between UACA2 and UAvicK, although differences between these strains existed. It was established that GbpB depletion impaired initial phases of sucrose-dependent biofilm formation, while exogenous native GbpB partially restored the biofilm phenotype. Several cellular traits were significantly affected by GbpB depletion, including altered cell shape, decreased autolysis, increased cell hydrophobicity, and sensitivity to antibiotics and osmotic and oxidative stresses. These data provide the first experimental evidence for GbpB participation in sucrose-dependent biofilm formation and in cell surface properties.
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Tabbassam Naheed Kauser, Abdul Ghafoor, Mohammad Sajjad, Zubaida Khanum, Bushra Nabi, and Hina Khan. "Frequency and Histopathological variants of Leiomyoma in Uterine Specimens in a Tertiary Care Hospital." Journal of Saidu Medical College, Swat 11, no. 1 (2021): 9–13. http://dx.doi.org/10.52206/jsmc.2021.11.1.9-13.
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Background: Leiomyoma is the commonest benign smooth muscle tumor of uterus. It also occur in other organs like gall bladder,skin, gasterointestinal tract etc. Leiomyosarcoma is a rare malignant counter part of leiomyoma.Objective: To see the frequency of histopathological variants of leiomyomas in uterine specimens in Southern District ofKhyber Pakhtunkhwa Pakistan.Material and Methods: This descriptive study was conducted in Department of Pathology, Bannu Medical College Bannu incollaboration with Government and Private Hospitals of the Southern District of KPK. The duration of study was seven years fromJanuary 2011 to December 2017. A total of 210 specimen of leiomyoma were included in this study. Inclusion criteria was allbiopsies of uterine leiomyomas of any age. Exclusion criteria wa autolysed and insufficient biopsy specimen. All biopsies werefixed over night in 10% buffered formalin, processed for histopathological slides preparation. Finally slides were prepared, labeledand reported by Histopathologist. . All the data was analysed in Statistical Package for Social Sciences (SPSS) version 20 forfrequency with percentages and mean with standard deviation.Results: In this study of 210 leiomyoma cases, the age range was from 25- 65 years. The commonest age group was 36-45 yearsfollowed by 25-35 years. The frequency of leiomyoma was 21.5% amongst the total uterine biopsy specimen. Histologically theusual leiomyoma was comprising of 176(83.80%) followed by hyalinised leiomyoma 11 (5.23%), myxoid leiomyoma 09(4.28%),lipoleiomyoma 05 (2.38%), cellular leiomyoma 04 (1.90%), shwannian leiomyoma 03 (1.42%) and one each of symplastic andangioleiomyoma 01(0.47%).Conclusin: Leiomyoma which is the commonest benign smooth muscle tumor of uterus have a number of histological variants. Inthis study usuall leiomyoma was the commonest variant followed by hyalinized leiomyoma, myxoid leiomyoma and lipoleiomyoma.It is important to separate various types of leiomyoma on histology to avoid confusion of misdiagnosis.Key WordsS: Histopathology. Fibroids. Leiomyoma variants. Hysterectomy. Myomectomy.
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Gottgens, Bertie. "Application of Single Cell Technologies to the Study of Hematopoiesis." Blood 134, Supplement_1 (2019): SCI—19—SCI—19. http://dx.doi.org/10.1182/blood-2019-132314.
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The Gottgens group uses a combination of experimental and computational approaches to study how transcription factor networks control the function of blood stem cells and how mutations that perturb such networks cause diseases. The group's current research focuses on (i) single cell genomics of early blood development, (ii) computer models of the transcriptional landscape of blood stem cell differentiation, (iii) transcriptional consequences of leukaemogenic mutations, and (iv) molecular characterization of human blood stem cell populations used in cell and gene therapy protocols. As requested by the session chair, this year's presentation will first provide an overview of single cell technologies, and how they are advancing our understanding of multiple facets of haematology research. This will include single cell molecular profiling, as well as single cell functional assays, and in particular also how a combination of the two allows a more precise definition of haematopoietic stem and progenitor cell types. The rest of the presentation will focus on our multidisciplinary work combining single cell molecular profiling, bioinformatics analysis and experimental/functional validation to study the normal haematopoiesis, and contrast this with 6 mouse models of pre-leukaemic disease. Comprehensive bioinformatics analysis reveals not only qualitative changes in cellular abundance, but also pinpoints the underlying molecular changes that are most likely driving the early stages of malignant disease. An overarching theme will be how single cell landscapes allow us to move seamlessly between different scales of biological investigation, from the molecular to the cellular and whole tissue scale. Finally, extrapolation to human patient data demonstrates disease relevance of gene sets identified from comparative analysis of single cell transcriptional landscapes in mouse models. Disclosures Gottgens: Astra Zeneca: Research Funding; GSK: Research Funding; Novo Nordisk: Consultancy, Research Funding; Autolus: Consultancy, Research Funding.
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O’Rourke, Katherine I., Timothy V. Baszler, Janice M. Miller, Terry R. Spraker, Ingrid Sadler-Riggleman, and Donald P. Knowles. "Monoclonal Antibody F89/160.1.5 Defines a Conserved Epitope on the Ruminant Prion Protein." Journal of Clinical Microbiology 36, no. 6 (1998): 1750–55. http://dx.doi.org/10.1128/jcm.36.6.1750-1755.1998.
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The transmissible spongiform encephalopathies are a heterogeneous group of fatal neurodegenerative disorders occurring in humans, mink, cats, and ruminant herbivores. The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests with standardized reagents. Postmortem diagnosis is performed by histologic examination of brain sections from affected animals. The histopathological criteria for transmissible spongiform encephalopathies include gliosis, astrocytosis, neuronal degeneration, and spongiform change. These lesions vary in intensity and anatomic location depending on the host species and genetics, stage of disease, and infectious agent source. Diagnosis by histopathology alone may be ambiguous in hosts with early cases of disease and impossible if the tissue is autolyzed. Deposition of the prion protein (an abnormal isoform of a native cellular sialoglycoprotein) in the central nervous system is a reliable marker for infection, and immunohistochemical detection of this marker is a useful adjunct to histopathology. In the present paper we describe monoclonal antibody (MAb) F89/160.1.5, which reacts with prion protein in tissues from sheep, cattle, mule deer, and elk with naturally occurring transmissible spongiform encephalopathies. This MAb recognizes a conserved epitope on the prion protein in formalin-fixed, paraffin-embedded sections after hydrated autoclaving. MAb F89/160.1.5 will be useful in diagnostic and pathogenesis studies of the transmissible spongiform encephalopathies in these ruminant species.
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Wu, Hui, Zhi-min Li, Li Zhou, and Qin Ye. "Improved Succinic Acid Production in the Anaerobic Culture of an Escherichia coli pflB ldhA Double Mutant as a Result of Enhanced Anaplerotic Activities in the Preceding Aerobic Culture." Applied and Environmental Microbiology 73, no. 24 (2007): 7837–43. http://dx.doi.org/10.1128/aem.01546-07.
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ABSTRACT Escherichia coli NZN111 is a pflB ldhA double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain.
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Weisburg, J. H., M. Curcio, P. C. Caron, et al. "The multidrug resistance phenotype confers immunological resistance." Journal of Experimental Medicine 183, no. 6 (1996): 2699–704. http://dx.doi.org/10.1084/jem.183.6.2699.
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Multidrug resistance (MDR), which is due, in part, to the overexpression of P-glycoprotein, confers resistance to a variety of natural product chemotherapeutic agents such as daunorubicin, vincristine, and colchicine. RV+ cells are a P-glycoprotein overexpressing variant of the HL60 myeloid leukemia cell line. In addition to classic MDR, RV+ cells displayed relative resistance to complement-mediated cytotoxicity with both immunoglobulin G and M antibodies against different cell surface antigens, but not to antibody-dependent cellular cytotoxicity and lymphokine-activated killing. Complement resistance was reversed both by treatment with verapamil and with specific monoclonal antibodies (mAbs) capable of binding to P-glycoprotein and blocking its function. To further confirm that the resistance of RV+ cells was not a consequence of the selection of the cells on vincristine, a second system involving P-glycoprotein infectants was also investigated. K562 cells infected with the MDR1 gene, which were never selected on chemotherapeutic drugs, also displayed relative resistance to complement-mediated cytotoxicity. This MDR1 infection-induced resistance was also reversed by mAbs that bind to P-glycoprotein. Therefore, the MDR phenotype as mediated by P-glycoprotein provides resistance to complement-mediated cytotoxicity. The increased intracellular pH and the decreased membrane potential due to the MDR phenotype may result in abnormal membrane attack complex function. This observation may have implications for the possible mechanisms of action of P-glycoprotein and for a possible physiologic role for P-glycoprotein in protection against complement-mediated autolysis.
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Ardeshna, Kirit, Maria A. V. Marzolini, Wendy Osborne, et al. "Study of AUTO3, the First Bicistronic Chimeric Antigen Receptor (CAR) Targeting CD19 and CD22, Followed By Anti-PD1 Consolidation in Patients with Relapsed/Refractory (r/r) Diffuse Large B Cell Lymphoma (DLBCL): Alexander Study." Blood 132, Supplement 1 (2018): 1679. http://dx.doi.org/10.1182/blood-2018-99-119197.
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Abstract Introduction CAR T-cell therapies directed against CD19 have shown significant activity in patients with r/r DLBCL; however, a significant number of patients relapse due to target antigen loss or upregulation of PDL1. In this study, we evaluate the safety and efficacy of AUTO3, a CAR T-cell therapy designed to target CD19 and CD22 simultaneously in order to reduce the likelihood of relapse due to antigen loss, followed by 3 doses of anti-PD1 monoclonal antibody pembrolizumab (pembro) consolidation treatment. Methods & Patients We constructed a novel bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. Antigen binding domains were humanized and both CARs are in "2nd generation" format (incorporating an OX40 co-stimulatory domain for the CD19 CAR and a 41BB for the CD22 CAR). The performance of the CD22 CAR was optimized by incorporating a novel pentameric spacer. The cell product was manufactured on a semi-automated and closed process using CliniMACS® Prodigy (Miltenyi Biotec). Patients (≥18 years) with r/r DLBCL not otherwise specified (NOS) or transformed from indolent histology; have chemorefractory disease or relapse after at least 2 prior therapies or autologous stem cell transplantation (ASCT); Eastern Cooperative Oncology Group performance status <2, adequate renal, hepatic, cardiac function, and an absolute lymphocyte count ≥0.5 x 10e9/L are eligible. Patients with CNS disease, prior allogeneic stem cell transplant, serious toxicity from prior CD19 or CD22 directed therapy are excluded, as well as patients with any contraindication to receiving pembro. Bridging therapy for disease control is allowed during the manufacturing period. All patients receive lymphodepletion with 30 mg/m2/day fludarabine and 300 mg/m2/day cyclophosphamide for 3 days prior to AUTO3 infusion. Three dose levels are being explored (50 x 10e6; 150 x 10e6, and 300 x 10e6 transduced CAR T cells). The first 3 patients in lowest dose cohort receive AUTO3 alone; subsequently, all patients receive AUTO3 followed by 3 doses of pembro 200 mg given every 3 weeks. The primary endpoint of Phase I is frequency of dose-limiting toxicities (DLTs) and grade (Gr) 3‒5 toxicity; key efficacy endpoints include overall response rate (ORR) based on Lugano classification, duration of response, and overall survival, as well as biomarker endpoints such as the level of AUTO3 cells in blood and duration of B-cell aplasia. Results As of the data cut-off date (July 20, 2018), 6 patients have been enrolled and dosed on study: 3 with AUTO3 alone and 2 with AUTO3 followed by pembro (1 patient is awaiting pembro dosing). All patients had product successfully manufactured and received 50 x 10e6 cells. The transduction efficiency was 17% (range 16‒29%). Median age was 35 years (range 28‒60), median prior lines of treatment was 3 (range 2‒4), 1 patient (17%) had prior ASCT, 4 patients (67%) had chemorefractory disease. Four patients had DLBCL NOS at initial diagnosis; 2 patients had transformed DLBCL from marginal zone and follicular lymphoma, respectively. Five patients had a minimum of 4 weeks' follow up and were evaluable for safety and efficacy analysis. No AUTO3-related deaths and no DLTs were observed. The most common Gr ≥3 adverse events were neutropenia in 5 patients (100%), platelet count decreased in 2 patients (40%), and hypophosphatemia in 2 patients (40%). One patient (20%) experienced Gr 1 cytokine release syndrome (CRS); no Gr 2 or higher CRS was observed. One patient (20%) who received AUTO3 alone had Gr 3 neurotoxicity that fully recovered. No immune adverse events related to pembro were observed and no patients required ICU admission. Four of the 5 patients had a response with ORR of 80% (95% CI 28.4‒99.5%). Two patients had a CR (40%; 95% CI 5.3‒85.3%) and continue to be in CR at the time of data cut off, with longest follow up of 3 months. CAR T-cell expansion was seen consistently in all patients. Conclusions This first study of simultaneously targeting CD19 and CD22 with a novel bicistronic CAR, AUTO3, demonstrates a manageable safety profile in combination with pembro. Early efficacy, even at the lowest dose of 50 x 10e6 cells, is promising with an ORR of 80% and 2 patients attaining a CR. The ALEXANDER study continues to enroll patients with AUTO3 followed by pembro. Updated follow up and additional patient data at higher dose levels, as well as cellular kinetics, relevant product characteristics, and biomarkers, will be presented. Disclosures Ardeshna: Celgene: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Osborne:Novartis: Other: Travel to conference; Pfizer: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy; Servier: Consultancy; Celgene: Consultancy. Al-Hajj:Autolus Ltd: Equity Ownership; Autolus Ltd: Employment. Thomas:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Faulkner:Autolus Ltd: Employment, Equity Ownership; GlaxoSmithKline: Equity Ownership. Pule:Autolus Ltd: Employment, Equity Ownership, Other: Salary contribution paid for by Autolus, Research Funding; University College London: Patents & Royalties: Patent with rights to Royalty share through UCL. Peddareddigari:Autolus Therapeutics plc: Employment; Autolus Therapeutics plc: Patents & Royalties; Autolus Therapeutics plc: Equity Ownership. Khokhar:Autolus Ltd: Employment; Autolus Ltd: Equity Ownership.
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Zeng, Jingmin, Rachael A. Dunlop, Kenneth J. Rodgers, and Michael J. Davies. "Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols." Biochemical Journal 398, no. 2 (2006): 197–206. http://dx.doi.org/10.1042/bj20060019.
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Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259–269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine–lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.
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Wallace, R. J., and Carol A. McPherson. "Factors affecting the rate of breakdown of bacterial protein in rumen fluid." British Journal of Nutrition 58, no. 2 (1987): 313–23. http://dx.doi.org/10.1079/bjn19870098.
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1. The cellular proteins ofButyrivibrio jibrisolvens, Lactobacillus casei, Megasphaera elsdenii, Selenomonas ruminantiumandStreptococcus boviswere labelled by growth in the presence of L-[14C]leucine, and the breakdown of labelled protein was measured in incubations of these bacteria with rumen fluid to which unlabelled 5 mM-L-leucine was added. The rate of protein breakdown was estimated from the rate of release of radioactivity into acid-soluble material.2. Protein breakdown occurred at different rates in different species. The mean rates for B.fibrisolvens, L. casei, M. elsdenii, Sel. ruminantiumandStr. boviswere 28.6, 18.1, 17.7, 10.5 and 5.3% /h respectively in samples of strained rumen fluid (SRF) with different protozoal populations. Rates of 3% /h or less were found in SRF from ciliate-free sheep or in faunated SRF from which protozoa had been removed by centrifugation. Further removal of mixed rumen bacteria had little effect. Suspensions of washed protozoa degraded bacterial protein at rates which were of the same order as those found in SRF.3. The rate of breakdown of bacterial protein in different samples of SRF tended to increase as the numbers of small entodiniomorphid protozoa increased. The numbers of larger entodiniomorphs and holotrichs had no obvious influence on this rate.4. Autoclaved and u.v.-treated bacteria were generally no different from live bacteria in their susceptibility to breakdown in SRF from faunated sheep, indicating that endogenous protein turnover was not a significant cause of bacterial protein catabolism.5. The rate of bacterial protein breakdown was unrelated to the proteolytic activity of SRF.6. It was concluded that predation by small protozoa is by far the most important cause of bacterial protein turnover in the rumen, with autolysis, other lytic factors and endogenous proteolysis being of minor importance.