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1

Pang, Qianqian, Qingai Lin, Di Wang, Zhenghao Sun, and Junfang Wang. "Molecular characterization of the Yp11.2 region deletion in the Chinese Han population." International Journal of Legal Medicine 135, no. 4 (April 26, 2021): 1351–58. http://dx.doi.org/10.1007/s00414-021-02596-x.

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AbstractThe Y chromosome is male-specific and is important for spermatogenesis and male fertility. However, the Y chromosome is poorly characterized due to massive palindromes and inverted repeats, which increase the likelihood of genomic rearrangements, resulting in short tandem repeats on the Y chromosome or long fragment deletions. The present study reports a large-scale (2.573~2.648 Mb) deletion in the Yp11.2 region in a Chinese population based on the analysis of 34 selected Y-specific sequence-tagged sites and subsequent sequencing of the breakpoint junctions on the Y chromosome from 5,068,482–5,142,391 bp to 7,715,462–7,716,695 bp. The results of sequence analysis indicated that the deleted region included part or all of the following five genes: PCDH11Y, TSPY, AMELY, TBL1Y, and RKY. These genes are associated with spermatogenesis or amelogenesis and various other processes; however, specific physiological functions and molecular mechanisms of these genes remain unclear. Notably, individuals with this deletion pattern did not have an obvious pathological phenotype but manifested some degree of amelogenesis imperfecta.
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2

Park, Joon Young, Miyuki Yamatani, Souhei Wadano, Yasuhiro Takagi, Kohsuke Honda, Takeshi Omasa, and Hisao Ohtake. "Effects of palindrome structure on Dhfr amplification in Chinese hamster ovary cells." Process Biochemistry 45, no. 12 (December 2010): 1845–51. http://dx.doi.org/10.1016/j.procbio.2009.11.019.

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3

Park, Joon Young, Yasuhiro Takagi, Miyuki Yamatani, Kohsuke Honda, Takeshi Omasa, and Hisao Ohtake. "Effects of palindrome structure on dihydrofolate reductase gene amplification in Chinese hamster ovary cells." Journal of Bioscience and Bioengineering 108 (November 2009): S9. http://dx.doi.org/10.1016/j.jbiosc.2009.08.035.

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4

Taghian, Danielle G., Heather Hough, and Jac A. Nickoloff. "Biased Short Tract Repair of Palindromic Loop Mismatches in Mammalian Cells." Genetics 148, no. 3 (March 1, 1998): 1257–68. http://dx.doi.org/10.1093/genetics/148.3.1257.

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Abstract Mismatch repair of palindromic loops in the presence or absence of single-base mismatches was investigated in wild-type and mismatch-binding defective mutant Chinese hamster ovary cells. Recombination intermediates with a maximum heteroduplex DNA (hDNA) region of 697 bp contained a centrally located, phenotypically silent 12-base palindromic loop mismatch, and/or five single-base mismatches. In wild-type cells, both loops and single-base mismatches were efficiently repaired (80–100%). When no other mismatches were present in hDNA, loops were retained with a 1.6–1.9:1 bias. However, this bias was eliminated when single-base mismatches were present, perhaps because single-base mismatches signal nick-directed repair. In the multiple marker crosses, most repair tracts were long and continuous, with preferential loss of markers in cis to proximal nicks, consistent with nicks directing most repair in this situation. However, ~25% of repair tracts were discontinuous as a result of loop-specific repair, or from segregation or short tract repair of single-base mismatches. In mutant cells, single-base mismatches were repaired less frequently, but the loop was still repaired efficiently and with bias toward loop retention, indicating that the defect in these cells does not affect loop-specific repair. Repair tracts in products from mutant cells showed a wide variety of mosaic patterns reflecting short regions of repair and segregation consistent with reduced nick-directed repair. In mutant cells, single-base mismatches were repaired more efficiently in the presence of the loop than in its absence, a likely consequence of corepair initiated at the loop.
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5

Leu, T. H., and J. L. Hamlin. "Activation of a mammalian origin of replication by chromosomal rearrangement." Molecular and Cellular Biology 12, no. 6 (June 1992): 2804–12. http://dx.doi.org/10.1128/mcb.12.6.2804.

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The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.
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6

Leu, T. H., and J. L. Hamlin. "Activation of a mammalian origin of replication by chromosomal rearrangement." Molecular and Cellular Biology 12, no. 6 (June 1992): 2804–12. http://dx.doi.org/10.1128/mcb.12.6.2804-2812.1992.

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The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.
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7

Lu, Wenwei, Zhangming Pei, Mengning Zang, Yuan-kun Lee, Jianxin Zhao, Wei Chen, Hongchao Wang, and Hao Zhang. "Comparative Genomic Analysis of Bifidobacterium bifidum Strains Isolated from Different Niches." Genes 12, no. 10 (September 25, 2021): 1504. http://dx.doi.org/10.3390/genes12101504.

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The potential probiotic benefits of Bifidobacterium bifidum have received increasing attention recently. We used comparative genomic analysis to explore the differences in the genome and the physiological characteristics of B. bifidum isolated from the fecal samples of Chinese adults and infants. The relationships between genotypes and phenotypes were analyzed to assess the effects of isolation sources on the genetic variation of B. bifidum. The phylogenetic tree results indicated that the phylogeny of B. bifidum may be related to the geographical features of its isolation source. B. bifidum was found to have an open pan-genome and a conserved core genome. The genetic diversity of B. bifidum is mainly reflected in carbohydrate metabolism- and immune/competition-related factors, such as the glycoside hydrolase gene family, bacteriocin operons, antibiotic resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Additionally, the type III A CRISPR-Cas system was discovered in B. bifidum for the first time. B. bifidum strains exhibited niche-specific characteristics, and the results of this study provide an improved understanding of the genetics of this species.
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8

HUANG, Chiu-Jung, Shinn-Chih WU, and Kong-Bung CHOO. "Transcriptional modulation of the pre-implantation embryo-specific Rnf35 gene by the Y-box protein NF-Y/CBF." Biochemical Journal 387, no. 2 (April 5, 2005): 367–75. http://dx.doi.org/10.1042/bj20041364.

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Maternal-to-zygotic transition of a fertilized egg and the subsequent pre-implantation development of the embryo involve zygotic genome activation and reprogramming of gene expression. The goal of the present study is to establish a model suitable for the characterization of transcriptional modulation of mammalian pre-implantation development. Rnf35 is a mouse RING-finger protein gene that is temporally transcribed in the early embryo, but is permanently silenced before the blastocyst stage of development. We first show that the Chinese-hamster ovary-K1 cells are unique in supporting Rnf35 promoter activities in transient transfection assays. Using the permissive Chinese-hamster ovary-K1 cell line, we show that Rnf35 transcription is driven by an Inr (initiator) core promoter element in the absence of a TATA box; the Inr promoter function is confirmed by direct microinjection of mouse one-cell embryos. This is the first demonstration of the involvement of an Inr core promoter element in transcription in pre-implantation development. We show that the Rnf35 promoter is regulated by three obligatory Y-box (CCAAT-box) elements: two Y boxes (YI and YII) located at −81 are coupled in a palindrome and act synergistically in contributing to Rnf35 transcription; the third Y box (YIII) is situated at −13, just upstream of the Inr element, and may be an integral part of the Inr function. Electrophoretic mobility-shift assays and competition experiments further reveal that the YI box is bound by the ubiquitous NF-Y (nuclear factor-Y)/CBF (CCAAT-binding factor) and that YII is targeted by an unidentified protein(s) that acts synergistically with the NF-Y. We suggest that the NF-Y, targeting at a Y-box sequence, may function as an important activator in transcriptional regulation of the Rnf35 gene in the pre-implantation embryo.
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9

Woźniakowski, Grzegorz, Natalia Mazur-Panasiuk, Marek Walczak, Małgorzata Juszkiewicz, Maciej Frant, and Krzysztof Niemczuk. "Attempts at the development of a recombinant African swine fever virus strain with abrogated EP402R, 9GL, and A238L gene structure using the CRISPR/Cas9 system." Journal of Veterinary Research 64, no. 2 (June 3, 2020): 197–205. http://dx.doi.org/10.2478/jvetres-2020-0039.

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AbstractIntroductionAfrican swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus’ mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain.Material and MethodsThe host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells.ResultsThe reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate.ConclusionTaking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.
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10

Dong, Yunpeng, Tao Peng, Weijing Wu, Donghui Tan, Xuezhong Liu, and Dinghua Xie. "Efficient introduction of an isogenic homozygous mutation to induced pluripotent stem cells from a hereditary hearing loss family using CRISPR/Cas9 and single-stranded donor oligonucleotides." Journal of International Medical Research 47, no. 4 (February 28, 2019): 1717–30. http://dx.doi.org/10.1177/0300060519829990.

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Background Heterozygous purinergic receptor p2x gene ( P2RX2) c.178G>T (p.V60L) mutations can lead to progressive hearing loss (HL) and increased susceptibility to noise. However, the underlying mechanisms remain unclear. A combination of human induced pluripotent stem cell (hiPSC) technology with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9-mediated gene editing may provide a promising tool to study gene function and treat hereditary deafness in humans. Methods hiPSC technology and CRISPR/Cas9-mediated gene editing were used to generate heterozygous and homozygous P2RX2 c.178G>T (p.V60L) cell models. Results We generated non-integrative hiPSCs from urine samples derived from three members of a large Chinese family carrying heterozygous P2RX2 c.178G>T mutations (designated P2RX2+/–) as a model to study P2RX2-mediated hereditary HL. Furthermore, we used CRISPR/Cas9 and single-stranded donor oligonucleotides to genetically establish homozygous P2RX2 c.178G>T hiPSCs (designated P2RX2–/–) from heterozygous patient-specific hiPSCs as a control to further study the pathological gene function. Conclusions Heterozygous and homozygous P2RX2-mutated hiPSC lines are good models to investigate the pathological mechanisms of P2RX2 mutations in HL pathogenesis. Our findings confirmed our hypothesis that it is feasible and convenient to introduce precise point mutations into genomic loci of interest to generate gene-mutated hiPSC models.
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11

Tang, Qiaomei. "From Talented Poet to Jealous Wife: Reimagining Su Hui in Late Tang Literary Culture." NAN Nü 22, no. 1 (June 8, 2020): 1–35. http://dx.doi.org/10.1163/15685268-00221p01.

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Abstract Su Hui was a late fourth century Chinese woman who is famed for her creation of brocade palindromic poems. Due to an account of her life story, attributed to the female emperor Wu Zetian, that highlighted her jealous disposition, Su Hui is remembered today primarily as a talented but jealous wife, which is in contrast with how she was viewed in the period prior to the Wu version. Tracing the genealogy of Su Hui’s narrative in pre-Tang and Tang literary and visual materials, this article demonstrates that the definitive version of Su Hui’s story is misattributed to Wu Zetian and, more importantly, that the image of this well-known figure of early medieval China underwent a transformation that reflects important aspects of Late Tang literary culture. In ‘boudoir lament’ poetry of the Southern Dynasties period, Su Hui is the stock image of a melancholy wife longing for her absent husband. In ‘frontier’ poetry of the Tang dynasty, she is a worrying wife concerned with her military husband fighting on the borderlands. It is in a Late Tang prose account misattributed to Wu Zetian that we finally see her as a jealous woman competing for her husband’s affections. The transformation of Su Hui’s image across three major literary genres over a period of half a millennium offers readers a window into the literary and cultural changes that took place in medieval China.
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Laanto, Elina, Janne J. Ravantti, and Lotta-Riina Sundberg. "Prophages and Past Prophage-Host Interactions Revealed by CRISPR Spacer Content in a Fish Pathogen." Microorganisms 8, no. 12 (December 2, 2020): 1919. http://dx.doi.org/10.3390/microorganisms8121919.

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The role of prophages in the evolution, diversification, or virulence of the fish pathogen Flavobacterium columnare has not been studied thus far. Here, we describe a functional spontaneously inducing prophage fF4 from the F. columnare type strain ATCC 23463, which is not detectable with commonly used prophage search methods. We show that this prophage type has a global distribution and is present in strains isolated from Finland, Thailand, Japan, and North America. The virions of fF4 are myoviruses with contractile tails and infect only bacterial strains originating from Northern Finland. The fF4 resembles transposable phages by similar genome organization and several gene orthologs. Additional bioinformatic analyses reveal several species in the phylum Bacteroidetes that host a similar type of putative prophage, including bacteria that are important animal and human pathogens. Furthermore, a survey of F. columnare Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers indicate a shared evolutionary history between F. columnare strains and the fF4 phage, and another putative prophage in the F. columnare strain ATCC 49512, named p49512. First, CRISPR spacer content from the two CRISPR loci (types II-C and VI-B) of the fF4 lysogen F. columnare ATCC 23463 revealed a phage terminase protein-matching spacer in the VI-B locus. This spacer is also present in two Chinese F. columnare strains. Second, CRISPR analysis revealed four F. columnare strains that contain unique spacers targeting different regions of the putative prophage p49512 in the F. columnare strain ATCC 49512, despite the geographical distance or genomovar of the different strains. This suggests a common ancestry for the F. columnare prophages and different host strains.
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Luciani, Paola, Cristiana Deledda, Fabiana Rosati, Susanna Benvenuti, Ilaria Cellai, Francesca Dichiara, Matteo Morello, et al. "Seladin-1 Is a Fundamental Mediator of the Neuroprotective Effects of Estrogen in Human Neuroblast Long-Term Cell Cultures." Endocrinology 149, no. 9 (May 22, 2008): 4256–66. http://dx.doi.org/10.1210/en.2007-1795.

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Estrogen exerts neuroprotective effects and reduces β-amyloid accumulation in models of Alzheimer’s disease (AD). A few years ago, a new neuroprotective gene, i.e. seladin-1 (for selective AD indicator-1), was identified and found to be down-regulated in AD vulnerable brain regions. Seladin-1 inhibits the activation of caspase-3, a key modulator of apoptosis. In addition, it has been demonstrated that the seladin-1 gene encodes 3β-hydroxysterol Δ24-reductase, which catalyzes the synthesis of cholesterol from desmosterol. We have demonstrated previously that in fetal neuroepithelial cells, 17β-estradiol (17βE2), raloxifene, and tamoxifen exert neuroprotective effects and increase the expression of seladin-1. The aim of the present study was to elucidate whether seladin-1 is directly involved in estrogen-mediated neuroprotection. Using the small interfering RNA methodology, significantly reduced levels of seladin-1 mRNA and protein were obtained in fetal neuroepithelial cells. Seladin-1 silencing determined the loss of the protective effect of 17βE2 against β-amyloid and oxidative stress toxicity and caspase-3 activation. A computer-assisted analysis revealed the presence of half-palindromic estrogen responsive elements upstream from the coding region of the seladin-1 gene. A 1490-bp region was cloned in a luciferase reporter vector, which was transiently cotransfected with the estrogen receptor α in Chinese hamster ovarian cells. The exposure to 17βE2, raloxifene, tamoxifen, and the soy isoflavones genistein and zearalenone increased luciferase activity, thus suggesting a functional role for the half-estrogen responsive elements of the seladin-1 gene. Our data provide for the first time a direct demonstration that seladin-1 may be considered a fundamental mediator of the neuroprotective effects of estrogen.
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Liu, Jingting, Mei Jiang, Haimei Chen, Yu Liu, Chang Liu, and Wuwei Wu. "Comparative genome analysis revealed gene inversions, boundary expansions and contractions, and gene loss in the Stemona sessilifolia (Miq.) Miq. chloroplast genome." PLOS ONE 16, no. 6 (June 18, 2021): e0247736. http://dx.doi.org/10.1371/journal.pone.0247736.

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Stemona sessilifolia (Miq.) Miq., commonly known as Baibu, is one of the most popular herbal medicines in Asia. In the Chinese Pharmacopoeia, Baibu has multiple authentic sources and there are many similar herbs sold as Baibu in herbal medicine markets. The existence of counterfeits of Baibu brings challenges to its identification. To assist in its accurate identification, we sequenced and analyzed the complete chloroplast genome of S. sessilifolia using next-generation sequencing technology. The genome was found to be 154,037 bp in length, possessing a typical quadripartite structure consisting of a pair of inverted repeats (IRs: 27,090 bp) separated by a large single copy (LSC: 81,949 bp) and a small single copy (SSC: 17,908 bp). A total of 112 unique genes were identified, including 80 protein-coding, 28 transfer RNA and four ribosomal RNA genes. In addition, 45 tandem, 27 forward, 23 palindromic and 104 simple sequence repeats were detected in the genome by repeated analysis. Compared with its counterfeits (Asparagus officinalis and Carludovica palmata) we found that IR expansion and SSC contraction events of S. sessilifolia resulted in two copies of the rpl22 gene in the IR regions and a partial duplication of the ndhF gene in the SSC region. An approximately 3-kb-long inversion was also identified in the LSC region, leading to the petA and cemA genes being presented in the complementary strand of the chloroplast DNA molecule. Comparative analysis revealed some highly variable regions, including trnF-GAA_ndhJ, atpB_rbcL, rps15_ycf1, trnG-UCC_trnR-UCU, ndhF_rpl32, accD_psaI, rps2_rpoC2, trnS-GCU_trnG-UCC, trnT-UGU_trnL-UAA and rps16_trnQ-UUG. Finally, gene loss events were investigated in the context of phylogenetic relationships. In summary, the complete plastome of S. sessilifolia will provide valuable information for the distinction between Baibu and its counterfeits and assist in elucidating the evolution of S. sessilifolia.
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Li, Chunjie, Jiabi Qian, Chuang Jiang, Ziping LI, and Hui Zhang. "Inherited GATA3 Variants Associated with Positive Minimal Residual Disease in Childhood B-ALL Via Autophagy-Induced Asparaginase Resistance." Blood 134, Supplement_1 (November 13, 2019): 654. http://dx.doi.org/10.1182/blood-2019-123575.

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Accumulating evidence has confirmed that inherited genetic variations play multi-dimensional roles in childhood acute lymphoblastic leukemia (ALL), i.e., leukemia susceptibility, treatment response, chemotherapy tolerance, and relapse. Germline variants at the GATA3 locus increase the risk of developing Philadelphia chromosome-like ALL (Ph-like ALL) and inferior outcomes in childhood B-ALL among European and American cohorts. However, the role of inherited GATA3 variants in Han Chinese children with ALL still remains unclear. To primarily identify the association of inherited GATA3 variants with treatment response, we retrospectively collected 273 childhood B-ALL blood samples after complete remission was achieved according to the Chinese Childhood Cancer Group ALL 2015. We then genotyped rs3824662 and rs3781093 in the GATA3 locus. The risk allele frequencies of rs3824662 and rs3781093 were 35.7% and 36.3%, respectively, consistent with the 1000 Genomes Project. Using a logistic regression model, we correlated the GATA3 genotype with minimal residual disease (MRD) level. In our single center, we found that GATA3 rs3824662 A allele and rs3781093 C allele statistically associated with positive MRD (the cut-off was >=0.01%, P=0.046 and 0.038, respectively). The A allele in rs3824662 and C allele in rs3781093 linked to 2-fold increase in the risk of MRD compared with their wildtype allele. To explore the biological functions of these two germline SNP variants, we first utilized luciferase reporter assay to determine the impact of GATA3 variants on its transcription activity. Interestingly, the rs3824662 risk A allele significantly increased enhancer activity, while the rs3781093 did not show any effect. We next genetically modified rs3824662 from wild-type C allele to A allele in the lymphoblastoid cell GM12878 using clustered regularly interspaced short palindromic repeats/associated 9 (CRISPR/Cas9) gene editing system. Compared with wildtype GM12878 cells, ~3-folds higher GATA3 transcription level was found in GM12878 cells with A/A or A/C genotype. Integrating the high risk of MRD and upregulated GATA3 expression, we proposed that GATA3 rs3824662 A allele might contribute to poor treatment response by promoting GATA3 transcription. To clarify the association of GATA3 expression with the sensitivity of ALL chemotherapeutic drugs, we retrieved GSE653 and GSE654 expression data for analysis and found that high GATA3 expression significantly correlated with L-asparaginase (L-Asp) and daunorubicin (DNR) resistance. To further confirm the correlation, we ectopically overexpressed GATA3 in B-ALL cell line (Nalm6) and only L-asp resistance was validated. L-asp resistance induced by GATA3 over-expression was rescued by GATA3 interference, consolidating the association between GATA3 and L-asp resistance in B-ALL cells. Next, we probed the mechanism of GATA3-mediated L-asp resistance in B-ALL. We analyzed the association between inherited GATA3 variants and L-asp allergy, and did not identify statistical significance. Meanwhile, we couldn't find the correlation between GATA3 and ASNS, suggesting that ASNS might not be the cause either. Intriguingly, we found GATA3 over-expression induced the activation of autophagy-related genes, BECN1 and ATG5, which was reported to be associated with L-Asp resistance. Tests on primary B-ALL samples further confirmed this findings. To further confirm the effect of GATA3 on autophagy, we cloned highly conserved sequence BECN1 or ATG5 promoter region into luciferase reporter constructs. The Over-expression of GATA3 dramatically increased luciferase activity compared with the corresponding empty vector (P = 0.0098 and 0.0114, respectively), indicating that GATA3 expression were functionally activate their transcription. Taken together, all these data indicated that higher GATA3 expression might induce L-Asp resistance in B-ALL cells via autophagy activation. In conclusion, we first identified that GATA3 rs3824662 associated with the risk of MRD in the childhood ALL cohorts of Han ethnicity. Mechanistic study showed that inherited GATA3 variants possibly contributed to L-Asp resistance via autophagy activation induced by promoting GATA3 enhancer activity, providing new insights into the rationale for the future development of combinational treatment of L-Asp and anti-autophagy regimen in ALL patients. Figure Disclosures No relevant conflicts of interest to declare.
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Liu, Yuejian, Feili Chen, Xutao Guo, Pengcheng Shi, Jie Zha, Zhiping Fan, Fen Huang, and Bing Xu. "Comparative Efficacy of Homoharringtonine Plus Cytarabine and Decitabine in Patients with MDS/AML." Blood 120, no. 21 (November 16, 2012): 4340. http://dx.doi.org/10.1182/blood.v120.21.4340.4340.

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Abstract Abstract 4340 Background: Myelodysplastic syndrome (MDS) is a malignant hematological disease that comprises a heterogeneous group of clonal hematopoietic stem and progenitor cell disorders, with peripheral cytopenias, bone marrow hypercellularity, high-risk of evolving into acute myeloid leukemia (AML). MDS/AML is a special refractory and palindromic AML characterized by poor therapeutic effects and low complete response rate, as well as high treatment-related complications and mortality. Patients with MDS/AML are often elders and represent more intolerance to routine or intensive chemotherapies. Homoharringtonine, an alkaloid found as the major active component in Chinese plants cephatotaxus fortuneif., has been widely used in AML since the 1970s in China. Decitabine, a hypomethylating agent, is active and has been approved for the treatment of myelodysplastic syndrome (MDS) in recent years. Objective: In order to compare the efficacy, toxicity and long-term prognosis of two chemotherapies HA (Homoharringtonine and cytarabine) and Decitabine regiment in MDS/AML. Methods: A total of 26 MDS/AML patients consisting of 14 males and 12 females were included in this study. They were randomly assigned to receive either HA (H 4mg.d−1,d1–3; A 100mg.m−2d−1, d1–7) or decitabine£.. 20mg.m−2d−1, d1–5£© The effect measures used were hazard ratios (HR) for overall survival (OS), progression-free survival (PFS) and freedom from first progression. Relative risks were used to analyse complete response rate, total response rate, treatment-related mortality and adverse events. A Log-rank test was used in survival analysis, and a Chi-square test was performed for other outcomes. Results: The complete remission (CR) rate with HA regimen according to MDS/AML criteria was 33% and 36% with decitabine (P>0.05). HA group had no lower total response rate than Decitabine group (53% versus 64%, P>0.05). The freedom from first progression in chemotherapy with HA regiment and decitabine was 20% and 18% (P>0.05), respectively. PFS was not statistically significantly longer for two comparators with HR was 0.41(95% confidence interval (CI) 0.09722 to 1.740). There was no statistically significant difference in OS between the HA group and decitabine group with HR was 0.799 (95% CI 0.2992 to 2.133); median survival: 300 days vs 291 days (P>0.05,95% confidence interval (CI) 0.6165 to 1.445). The treatment-related mortality was 13% with HA regimen versus 18% with decitabine at 3 weeks (P>0.05) and 40% with HA regiment versus 18% with decitabine at 3 months (P>0.05). The haematological toxicities and liver function lesion WHO grade III or IV were not significantly higher in the HA group than that in the decitabine group (P>0.05). The total secondary infection rates in all sections of chemotherapies were 58% and 19% (P=0.005) in the two groups, respectively. Secondary infection rate was significantly lower in the decitabine group than that in the HA group. Conclusions: This analysis showed that Homoharringtonine and cytarabine regiment in treating MDS/AML has a similar therapeutic effect and long-term benefit with decitabine, both regiments were associated with relatively safe and effective outcomes in patients with MDS/AML. However, HA regiment shows a higher risk of secondary infection than decitabine. Longer follow-up and further studies will evaluate prospectively the results of HA regiment versus decitabine in this setting. Disclosures: No relevant conflicts of interest to declare.
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Safari, Fatemeh, Safar Farajnia, Abbas Behzad Behbahani, Habib Zarredar, Mazyar Barekati-Mowahed, and Hesam Dehghani. "Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability." Biological Research 53, no. 1 (November 13, 2020). http://dx.doi.org/10.1186/s40659-020-00319-x.

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Abstract Background Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell in the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. Results Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. Conclusion These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.
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18

Hu, Xingming, Yongtao Cui, Guojun Dong, Anhui Feng, Danying Wang, Chunyan Zhao, Yu Zhang, et al. "Using CRISPR-Cas9 to generate semi-dwarf rice lines in elite landraces." Scientific Reports 9, no. 1 (December 2019). http://dx.doi.org/10.1038/s41598-019-55757-9.

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AbstractGenetic erosion refers to the loss of genetic variation in a crop. In China, only a few original landraces of rice (Oryza sativa) were used in breeding and these became the primary genetic background of modern varieties. Expanding the genetic diversity among Chinese rice varieties and cultivating high-yielding and high-quality varieties with resistance to different biotic and abiotic stresses is critical. Here, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9(Cas9) genome editing system to edit Semi-Dwarf1 (SD1) in the elite landraces Kasalath and TeTePu (TTP), which contain many desired agronomic traits such as tolerance to low phosphorous and broad-spectrum resistance to several diseases and insects. Mutations of SD1 confer shorter plant height for better resistance to lodging. Field trials demonstrated that the yield of the new Kasalath and TTP mutant lines was better than that of the wild type under modern cultivation and that the lines maintained the same desirable agronomic characteristics as their wild-type progenitors. Our results showed that breeding using available landraces in combination with genomic data of different landraces and gene-editing techniques is an effective way to relieve genetic erosion in modern rice varieties.
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Chua, Choon-Guan, and Bernard Yu-Hor Thong. "Inflammatory Arthritis Among Military Servicemen From a Rheumatology Center in Singapore." Military Medicine, June 30, 2021. http://dx.doi.org/10.1093/milmed/usab246.

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ABSTRACT Introduction Musculoskeletal disorders are one of the most common reasons military servicemen seek medical care during their line of duty. This study aims to review the clinical profile and outcomes of military personnel with inflammatory arthritis (IA) referred to a specialist rheumatology center in Singapore. Materials and Methods Consecutive new case referrals from the Singapore Armed Forces medical centers during the study period January 1, 2010, to December 31, 2019, were retrospectively studied. Results There were 123 referrals, comprising 112 (91.1%) males, with the majority being Chinese (110, 89.4%). The mean age was 25.5 ± 11.1 years. The most common diagnoses were gout (including chronic tophaceous gout; 34, 27.6%), spondyloarthritis (18, 14.6%), palindromic rheumatism (8, 6.5%), rheumatoid arthritis (4, 3.3%), and juvenile idiopathic arthritis (4, 3.3%). Among servicemen with gout, all were male, the majority (31, 91.3%) were Chinese, and mean age was 34.1 ± 8.8 years. Mean body mass index (BMI) was 27.5 ± 3.9 kg/m2, of which 41.2% had moderate-risk and 47.1% high-risk BMI for cardiovascular disease and diabetes mellitus (DM). Comorbidities included hyperlipidemia (14), hypertension (6), and type 2 DM (3). Urate lowering therapy was initiated in 27 (79.4%) patients, comprising allopurinol (85.2%), probenecid (11.1%), and their combination (3.7%). One patient developed allopurinol-induced hepatitis; none had severe cutaneous adverse reactions. Among the remaining patients with IA, conventional synthetic disease-modifying antirheumatic drugs (DMARDs) used were sulfasalazine (8), methotrexate (4), hydroxychloroquine (4), and leflunomide (2). Biologic DMARDs used in five patients comprised adalimumab (3) and golimumab (2). Conclusion Servicemen with IA and good functional status can still be physically fit and deployable into certain combat and service support vocations. This will optimize manpower resources in military organizations with a shrinking young workforce.
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Zhang, Fushun, Xiangzhi Meng, Michael B. Townsend, Panayampalli Subbian Satheshkumar, and Yan Xiang. "Identification of CP77 as the Third Orthopoxvirus SAMD9 and SAMD9L Inhibitor with Unique Specificity for a Rodent SAMD9L." Journal of Virology 93, no. 12 (March 27, 2019). http://dx.doi.org/10.1128/jvi.00225-19.

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ABSTRACTOrthopoxviruses (OPXVs) have a broad host range in mammalian cells, but Chinese hamster ovary (CHO) cells are nonpermissive for vaccinia virus (VACV). Here, we revealed a species-specific difference in host restriction factor SAMD9L as the cause for the restriction and identified orthopoxvirus CP77 as a unique inhibitor capable of antagonizing Chinese hamster SAMD9L (chSAMD9L). Two known VACV inhibitors of SAMD9 and SAMD9L (SAMD9&L), K1 and C7, can bind human and mouse SAMD9&L, but neither can bind chSAMD9L. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 knockout of chSAMD9L from CHO cells removed the restriction for VACV, while ectopic expression of chSAMD9L imposed the restriction for VACV in a human cell line, demonstrating that chSAMD9L is a potent restriction factor for VACV. In contrast to K1 and C7, cowpox virus CP77 can bind chSAMD9L and rescue VACV replication in cells expressing chSAMD9L, indicating that CP77 is yet another SAMD9L inhibitor but has a unique specificity for chSAMD9L. Binding studies showed that the N-terminal 382 amino acids of CP77 were sufficient for binding chSAMD9L and that both K1 and CP77 target a common internal region of SAMD9L. Growth studies with nearly all OPXV species showed that the ability of OPXVs to antagonize chSAMD9L correlates with CP77 gene status and that a functional CP77 ortholog was maintained in many OPXVs, including monkeypox virus. Our data suggest that a species-specific difference in rodent SAMD9L poses a barrier for cross-species OPXV infection and that OPXVs have evolved three SAMD9&L inhibitors with different specificities to overcome this barrier.IMPORTANCESeveral OPXV species, including monkeypox virus and cowpox virus, cause zoonotic infection in humans. They are believed to use wild rodents as the reservoir or intermediate hosts, but the host or viral factors that are important for OPXV host range in rodents are unknown. Here, we showed that the abortive replication of several OPXV species in a Chinese hamster cell line was caused by a species-specific difference in the host antiviral factor SAMD9L, suggesting that SAMD9L divergence in different rodent species poses a barrier for cross-species OPXV infection. While the Chinese hamster SAMD9L could not be inhibited by two previously identified OPXV inhibitors of human and mouse SAMD9&L, it can be inhibited by cowpox virus CP77, indicating that OPXVs encode three SAMD9&L inhibitors with different specificities. Our data suggest that OPXV host range in broad rodent species depends on three SAMD9&L inhibitors with different specificities.
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Wanga, Vincent Okelo, Xiang Dong, Millicent Akinyi Oulo, Elijah Mbandi Mkala, Jia-Xin Yang, Guy Eric Onjalalaina, Moses Kirega Gichua, et al. "Complete Chloroplast Genomes of Acanthochlamys bracteata (China) and Xerophyta (Africa) (Velloziaceae): Comparative Genomics and Phylogenomic Placement." Frontiers in Plant Science 12 (June 14, 2021). http://dx.doi.org/10.3389/fpls.2021.691833.

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Acanthochlamys P.C. Kao is a Chinese endemic monotypic genus, whereas XerophytaJuss. is a genus endemic to Africa mainland, Arabian Peninsula and Madagascar with ca.70 species. In this recent study, the complete chloroplast genome of Acanthochlamys bracteata was sequenced and its genome structure compared with two African Xerophyta species (Xerophyta spekei and Xerophyta viscosa) present in the NCBI database. The genomes showed a quadripartite structure with their sizes ranging from 153,843 bp to 155,498 bp, having large single-copy (LSC) and small single-copy (SSC) regions divided by a pair of inverted repeats (IR regions). The total number of genes found in A. bracteata, X. spekei and X. viscosa cp genomes are 129, 130, and 132, respectively. About 50, 29, 28 palindromic, forward and reverse repeats and 90, 59, 53 simple sequence repeats (SSRs) were found in the A. bracteata, X. spekei, and X. viscosa cp genome, respectively. Nucleotide diversity analysis in all species was 0.03501, Ka/Ks ratio average score was calculated to be 0.26, and intergeneric K2P value within the Order Pandanales was averaged to be 0.0831. Genomic characterization was undertaken by comparing the genomes of the three species of Velloziaceae and it revealed that the coding regions were more conserved than the non-coding regions. However, key variations were noted mostly at the junctions of IRs/SSC regions. Phylogenetic analysis suggests that A. bracteata species has a closer genetic relationship to the genus Xerophyta. The present study reveals the complete chloroplast genome of A. bracteata and gives a genomic comparative analysis with the African species of Xerophyta. Thus, can be useful in developing DNA markers for use in the study of genetic variabilities and evolutionary studies in Velloziaceae.
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Sosnovtsev, Stanislav V., Carlos Sandoval-Jaime, Gabriel I. Parra, Christine M. Tin, Ronald W. Jones, Jo Soden, Donna Barnes, Jim Freeth, Alvin W. Smith, and Kim Y. Green. "Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells." mBio 8, no. 1 (February 14, 2017). http://dx.doi.org/10.1128/mbio.00031-17.

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ABSTRACTThe Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin) were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.IMPORTANCEVesiviruses, such as San Miguel sea lion virus and feline calicivirus, are typically associated with infection in animal hosts. Following the accidental infection of a laboratory worker with San Miguel sea lion virus, a related virus was isolated in cell culture and named Hom-1. In this study, we found that Hom-1 could be propagated in a number of human cell lines, making it the first calicivirus to replicate efficiently in cultured human cells. Screening of a library of human cell surface membrane proteins showed that the virus could utilize human junctional adhesion molecule 1 as a receptor to enter cells and initiate replication. The Hom-1 virus presents a new system for the study of calicivirus biology and species specificity.
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van Wijk, Xander M., Simon Döhrmann, Björn M. Hallström, Shangzhong Li, Bjørn G. Voldborg, Brandon X. Meng, Karen K. McKee, et al. "Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion." mBio 8, no. 1 (January 10, 2017). http://dx.doi.org/10.1128/mbio.02128-16.

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ABSTRACT To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2β1γ1/laminin-α2β2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens. IMPORTANCE Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin α2 subunit. This finding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs. IMPORTANCE Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin α2 subunit. This finding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs.
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Yang, Heyu, Liqiang Wang, Haimei Chen, Mei Jiang, Wuwei Wu, Shengyu Liu, Jiehua Wang, and Chang Liu. "Phylogenetic analysis and development of molecular markers for five medicinal Alpinia species based on complete plastome sequences." BMC Plant Biology 21, no. 1 (September 22, 2021). http://dx.doi.org/10.1186/s12870-021-03204-1.

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Abstract Background Alpinia species are widely used as medicinal herbs. To understand the taxonomic classification and plastome evolution of the medicinal Alpinia species and correctly identify medicinal products derived from Alpinia species, we systematically analyzed the plastome sequences from five Alpinia species. Four of the Alpinia species: Alpinia galanga (L.) Willd., Alpinia hainanensis K.Schum., Alpinia officinarum Hance, and Alpinia oxyphylla Miq., are listed in the Chinese pharmacopeia. The other one, Alpinia nigra (Gaertn.) Burtt, is well known for its medicinal values. Results The four Alpinia species: A. galanga, A. nigra, A. officinarum, and A. oxyphylla, were sequenced using the Next-generation sequencing technology. The plastomes were assembled using Novoplasty and annotated using CPGAVAS2. The sizes of the four plastomes range from 160,590 bp for A. galanga to 164,294 bp for A. nigra, and display a conserved quadripartite structure. Each of the plastomes encodes a total of 111 unique genes, including 79 protein-coding, 28 tRNA, and four rRNA genes. In addition, 293–296 SSRs were detected in the four plastomes, of which the majority are mononucleotides Adenine/Thymine and are found in the noncoding regions. The long repeat analysis shows all types of repeats are contained in the plastomes, of which palindromic repeats occur most frequently. The comparative genomic analyses revealed that the pair of the inverted repeats were less divergent than the single-copy region. Analysis of sequence divergence on protein-coding genes showed that two genes (accD and ycf1) had undergone positive selection. Phylogenetic analysis based on coding sequence of 77 shared plastome genes resolves the molecular phylogeny of 20 species from Zingiberaceae. In particular, molecular phylogeny of four sequenced Alpinia species (A. galanga, A. nigra, A. officinarum, and A. oxyphylla) based on the plastome and nuclear sequences showed congruency. Furthermore, a comparison of the four newly sequenced Alpinia plastomes and one previously reported Alpinia plastomes (accession number: NC_048461) reveals 59 highly divergent intergenic spacer regions. We developed and validated two molecular markers Alpp and Alpr, based on two regions: petN-psbM and psaJ-rpl33, respectively. The discrimination success rate was 100 % in validation experiments. Conclusions The results from this study will be invaluable for ensuring the effective and safe uses of Alpinia medicinal products and for the exploration of novel Alpinia species to improve human health.
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