93 results on '"gene copy number variations"'
Search Results
2. Ten-year persistence and evolution of Plasmodium falciparum antifolate and anti-sulfonamide resistance markers pfdhfr and pfdhps in three Asian countries.
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Srisutham, Suttipat, Madmanee, Wanassanan, Kouhathong, Jindarat, Sutawong, Kreepol, Tripura, Rupam, Peto, Thomas J., van der Pluijm, Rob W., Callery, James J., Dysoley, Lek, Mayxay, Mayfong, Newton, Paul N., Pongvongsa, Tiengkham, Hongvanthong, Bouasy, Day, Nicholas P. J., White, Nicholas J., Dondorp, Arjen M., and Imwong, Mallika
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PLASMODIUM falciparum ,SINGLE nucleotide polymorphisms ,GENE amplification ,POLYMERASE chain reaction ,GENETIC mutation - Abstract
Background: The amplification of GTP cyclohydrolase 1 (pfgch1) in Plasmodium falciparum has been linked to the upregulation of the pfdhfr and pfdhps genes associated with resistance to the antimalarial drug sulfadoxine-pyrimethamine. During the 1990s and 2000s, sulfadoxine-pyrimethamine was withdrawn from use as first-line treatment in southeast Asia due to clinical drug resistance. This study assessed the temporal and geographic changes in the prevalence of pfdhfr and pfdhps gene mutations and pfgch1 amplification a decade after sulfadoxine-pyrimethamine had no longer been widely used. Methods: A total of 536 P. falciparum isolates collected from clinical trials in Thailand, Cambodia, and Lao PDR between 2008 and 2018 were assayed. Single nucleotide polymorphisms of the pfdhfr and pfdhps genes were analyzed using nested PCR and Sanger sequencing. Gene copy number variations of pfgch1 were investigated using real-time polymerase chain reaction assay. Results: Sequences of the pfdhfr and pfdhps genes were obtained from 96% (517/536) and 91% (486/536) of the samples, respectively. There were 59 distinct haplotypes, including single to octuple mutations. The two major haplotypes observed included IRNI-AGEAA (25%) and IRNL-SGKGA (19%). The sextuple mutation IRNL-SGKGA increased markedly over time in several study sites, including Pailin, Preah Vihear, Ratanakiri, and Ubon Ratchathani, whereas IRNI-AGEAA decreased over time in Preah Vihear, Champasak, and Ubon Ratchathani. Octuple mutations were first observed in west Cambodia in 2011 and subsequently in northeast Cambodia, as well as in southern Laos by 2018. Amplification of the pfgch1 gene increased over time across the region, particularly in northeast Thailand close to the border with Laos and Cambodia. Conclusion: Despite the fact that SP therapy was discontinued in Thailand, Cambodia, and Laos decades ago, parasites retained the pfdhfr and pfdhps mutations. Numerous haplotypes were found to be prevalent among the parasites. Frequent monitoring of pfdhfr and pfdhps in these areas is required due to the relatively rapid evolution of mutation patterns. [ABSTRACT FROM AUTHOR]
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- 2022
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3. Expanded genome-wide comparisons give novel insights into population structure and genetic heterogeneity of Leishmania tropica complex.
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Salloum, Tamara, Moussa, Rim, Rahy, Ryan, Al Deek, Jospin, Khalifeh, Ibrahim, El Hajj, Rana, Hall, Neil, Hirt, Robert P., and Tokajian, Sima
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LEISHMANIA mexicana ,GENE ontology ,CUTANEOUS leishmaniasis ,SINGLE nucleotide polymorphisms ,DNA copy number variations ,PRINCIPAL components analysis ,LEISHMANIA - Abstract
Leishmania tropica is one of the main causative agents of cutaneous leishmaniasis (CL). Population structures of L. tropica appear to be genetically highly diverse. However, the relationship between L. tropica strains genomic diversity, protein coding gene evolution and biogeography are still poorly understood. In this study, we sequenced the genomes of three new clinical L. tropica isolates, two derived from a recent outbreak of CL in camps hosting Syrian refugees in Lebanon and one historical isolate from Azerbaijan to further refine comparative genome analyses. In silico multilocus microsatellite typing (MLMT) was performed to integrate the current diversity of genome sequence data in the wider available MLMT genetic population framework. Single nucleotide polymorphism (SNPs), gene copy number variations (CNVs) and chromosome ploidy were investigated across the available 18 L. tropica genomes with a main focus on protein coding genes. MLMT divided the strains in three populations that broadly correlated with their geographical distribution but not populations defined by SNPs. Unique SNPs profiles divided the 18 strains into five populations based on principal component analysis. Gene ontology enrichment analysis of the protein coding genes with population specific SNPs profiles revealed various biological processes, including iron acquisition, sterols synthesis and drug resistance. This study further highlights the complex links between L. tropica important genomic heterogeneity and the parasite broad geographic distribution. Unique sequence features in protein coding genes identified in distinct populations reveal potential novel markers that could be exploited for the development of more accurate typing schemes to further improve our knowledge of the evolution and epidemiology of the parasite as well as highlighting protein variants of potential functional importance underlying L. tropica specific biology. Author summary: Human cutaneous leishmaniasis (CL) is a parasitic infection transmitted through the bite of an infected sandfly vector. Members of the Leishmania tropica complex are one of the main causative agents of CL in the Old World. Despite having varying phenotypical manifestations and degrees of severity, the genomic differences underlying the observed phenotypic diversity are not well understood. CL outbreaks in Lebanon, Syria and in Azerbaijan and neighbouring countries are evidence that leishmaniasis is still an important public health concern. Here, we explored unique genetic features in L. tropica genomes by comparing the gene variants and gene copy number variations, as well as chromosome ploidy, of two recent isolates collected during a CL outbreak in Lebanon and one historical isolate from Azerbaijan with 16 published L. tropica genomes. In silico MLMT was also performed to integrate the current diversity of genome sequence data in the wider geography-related genetic populations. This study further highlights the distinct population structure and genomic heterogeneity of the L. tropica species complex in the context of the parasite broad geographic distribution. Unique sequence features identified in distinct populations could be exploited for the development of more accurate typing schemes and highlight proteins of potential functional importance to gain new insights into L. tropica specific biology. [ABSTRACT FROM AUTHOR]
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- 2020
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4. High-quality reference genome of Fasciola gigantica: Insights into the genomic signatures of transposon-mediated evolution and specific parasitic adaption in tropical regions.
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Luo, Xier, Cui, Kuiqing, Wang, Zhiqiang, Li, Zhipeng, Wu, Zhengjiao, Huang, Weiyi, Zhu, Xing-Quan, Ruan, Jue, Zhang, Weiyu, and Liu, Qingyou
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GENE ontology ,FASCIOLA ,FASCIOLA hepatica ,LIVER flukes ,NEGLECTED diseases ,FLAVOBACTERIUM ,MIOCENE Epoch ,CLONORCHIS sinensis - Abstract
Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis, with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying effect. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 predicted excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions. Author summary: Fascioliasis is a neglected zoonotic tropical disease of humans, which reduces the productivity of animal industries, and imposes an economic burden of at least 3.2 billion dollars annually. Although there are four assemblies for F. hepatica and two assemblies for F. gigantica at NCBI, the inherent limited ability of short reads based assemblies made the completeness of genome sequences and the quality of gene annotation challenging. Here, we report the Pacbio assembly of reference genome for F. gigantica, and the quality of assembly and gene annotation are significantly improved compared with previous assemblies. Besides, we found the evidence of transposon-mediated evolution, especially for LINE insertions into gene regions between 41 Ma and 62 Ma, contributing to the speciation and adaption of the Fasciola ancestors. Furthermore, we identified F. gigantica specific gene duplication including 98 gene families, and 3300 protein-protein interactions between F. gigantica and the host in the small intestine and liver environment. These results illustrate the genomic and gene evolution of F. gigantica potentially shaping multiple parasitic characters. [ABSTRACT FROM AUTHOR]
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- 2021
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5. Distribution of insecticide resistance and mechanisms involved in the arbovirus vector Aedes aegypti in Laos and implication for vector control.
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Marcombe, Sébastien, Fustec, Bénédicte, Cattel, Julien, Chonephetsarath, Somesanith, Thammavong, Phoutmany, Phommavanh, Nothasin, David, Jean-Philippe, Corbel, Vincent, Sutherland, Ian W., Hertz, Jeffrey C., and Brey, Paul T.
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INSECTICIDE-treated mosquito nets ,INSECTICIDE resistance ,AEDES aegypti ,VECTOR control ,YELLOW fever ,CYTOCHROME P-450 - Abstract
Background: The yellow fever mosquito Aedes aegypti is the major vector of dengue, yellow fever, Zika, and Chikungunya viruses. Worldwide vector control is largely based on insecticide treatments but, unfortunately, vector control programs are facing operational challenges due to mosquitoes becoming resistant to commonly used insecticides. In Southeast Asia, resistance of Ae. aegypti to chemical insecticides has been documented in several countries but no data regarding insecticide resistance has been reported in Laos. To fill this gap, we assessed the insecticide resistance of 11 Ae. aegypti populations to larvicides and adulticides used in public health operations in the country. We also investigated the underlying molecular mechanisms associated with resistance, including target site mutations and detoxification enzymes putatively involved in metabolic resistance. Methods & results: Bioassays on adults and larvae collected in five provinces revealed various levels of resistance to organophosphates (malathion and temephos), organochlorine (DDT) and pyrethroids (permethrin and deltamethrin). Synergist bioassays showed a significant increased susceptibility of mosquitoes to insecticides after exposure to detoxification enzyme inhibitors. Biochemical assays confirmed these results by showing significant elevated activities of cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST) and carboxylesterases (CCE) in adults. Two kdr mutations, V1016G and F1534C, were detected by qPCR at low and high frequency, respectively, in all populations tested. A significant negative association between the two kdr mutations was detected. No significant association between kdr mutations frequency (for both 1534C and 1016G) and survival rate to DDT or permethrin (P > 0.05) was detected. Gene Copy Number Variations (CNV) were detected for particular detoxification enzymes. At the population level, the presence of CNV affecting the carboxylesterase CCEAE3A and the two cytochrome P450 CYP6BB2 and CYP6P12 were significantly correlated to insecticide resistance. Conclusions: These results suggest that both kdr mutations and metabolic resistance mechanisms are present in Laos but their impact on phenotypic resistance may differ in proportion at the population or individual level. Molecular analyses suggest that CNV affecting CCEAE3A previously associated with temephos resistance is also associated with malathion resistance while CNV affecting CYP6BB2 and CYP6P12 are associated with pyrethroid and possibly DDT resistance. The presence of high levels of insecticide resistance in the main arbovirus vector in Laos is worrying and may have important implications for dengue vector control in the country. Author summary: Aedes aegypti is the major vector of dengue in Laos and the control of this vector rely mainly on insecticide treatments. Compared to the neighboring countries, where resistance has been detected, there was no data on the distribution, the levels, and the mechanisms involved in the resistance in Laos. Laboratory bioassays showed that resistance to the currently used larvicides (temephos) and adulticides (pyrethroids) was present at different levels and distributed throughout the country. This may have important implications for dengue vector control in Laos. The mechanisms underlying the resistance were determined to be both metabolic and target site mutations (kdr) supporting results found in other countries. Several key detoxification enzyme genes were identified as potential candidates for metabolic resistance. This study provides a baseline on insecticide resistance in Laos and will help the Public Health authorities in designing more adapted vector control strategies. [ABSTRACT FROM AUTHOR]
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- 2019
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6. Genome-wide analysis identifies rare copy number variations associated with inflammatory bowel disease.
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Frenkel, Svetlana, Bernstein, Charles N., Sargent, Michael, Kuang, Qin, Jiang, Wenxin, Wei, John, Thiruvahindrapuram, Bhooma, Spriggs, Elizabeth, Scherer, Stephen W., and Hu, Pingzhao
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FALSE discovery rate ,INFLAMMATORY bowel diseases ,LOCUS (Genetics) ,MICROARRAY technology ,COMPUTATIONAL biology ,HUMAN genetics ,ETIOLOGY of diseases - Abstract
Background: Inflammatory bowel disease (IBD) is an idiopathic, chronic disorder of unclear etiology with an underlying genetic predisposition. Recent genome-wide association studies have identified more than 200 IBD susceptibility loci, but the causes of IBD remain poorly defined. We hypothesized that rare (<0.1% population frequency) gene copy number variations (CNVs) could play an important mechanism for risk of IBD. We aimed to examine changes in DNA copy number in a population-based cohort of patients with IBD and search for novel genetic risk factors for IBD. Methods: DNA samples from 243 individuals with IBD from the Manitoba IBD Cohort Study and 2988 healthy controls were analyzed using genome-wide SNP microarray technology. Three CNV calling algorithms were applied to maximize sensitivity and specificity of CNV detection. We identified IBD-associated genes affected by rare CNV from comparing the number of overlapping CNVs in IBD samples with the number of overlapping CNVs in controls for each gene. Results: 4,402 CNVs detected by two or three algorithms intersected 7,061 genes, in at least one analyzed sample. Four genes (e.g. DUSP22 and IP6K3) intersected by rare deletions and fourteen genes (e.g. SLC25A10, PSPN, GTF2F1) intersected by rare duplications demonstrated significant association with IBD (FDR-adjusted p-value < 0.01). Of these, ten genes were functionally related to immune response and intracellular signalling pathways. Some of these genes were also identified in other IBD related genome-wide association studies. These suggested that the identified genes may play a role in the risk of IBD. Conclusion: Our results revealed new genomic loci associated with IBD, which suggested the role of rare CNVs in IBD risk. [ABSTRACT FROM AUTHOR]
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- 2019
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7. Association of a rapidly selected 4.3kb transposon-containing structural variation with a P450-based resistance to pyrethroids in the African malaria vector Anopheles funestus.
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Mugenzi, Leon M. J., Tekoh, Theofelix A., Ntadoun, Stevia T., Chi, Achille D., Gadji, Mahamat, Menze, Benjamin D., Tchouakui, Magellan, Irving, Helen, Wondji, Murielle J., Weedall, Gareth D., Hearn, Jack, and Wondji, Charles S.
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ANOPHELES ,MALARIA ,PYRETHROIDS ,GENE expression ,MALARIA prevention - Abstract
Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance. Author summary: Anopheles funestus is an important malaria transmitting mosquito whose control relies on pyrethroid insecticides. The ability to control malaria transmitting mosquitoes is threatened by the emergence of insecticide resistance. Developing effective strategies to prevent, slow, or overcome resistance require an understanding of the underlying genetic mechanisms. In this study, we identified a transposon containing insertion in Anopheles funestus mosquitoes in Central/East Africa but absent in other regions. Tracking its distribution and frequency over the years revealed a strong selection with frequencies reaching fixation in less than 3 years. Phenotype/genotype association studies revealed a strong correlation between pyrethroid resistance and genetic variant genotype but negative correlation with plasmodium parasite infection in mosquitoes. These findings shed light on the often-overlooked role of structural variations in the evolution of insecticide resistance, offering valuable insights and tools for monitoring and addressing this critical issue in malaria control. [ABSTRACT FROM AUTHOR]
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- 2024
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8. Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1.
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Tulloch, Lindsay B., Tinti, Michele, Wall, Richard J., Weidt, Stefan K., Corpas- Lopez, Victoriano, Dey, Gourav, Smith, Terry K., Fairlamb, Alan H., Barrett, Michael P., and Wyllie, Susan
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CYTOCHROME P-450 ,LEISHMANIA donovani ,DEMETHYLASE ,VISCERAL leishmaniasis ,AMPHOTERICIN B ,PHYTOSTEROLS - Abstract
Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of L. donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway. Author summary: The antifungal drug, amphotericin B, is also used in the treatment of visceral leishmaniasis, a potentially lethal parasitic disease infecting the specialised immune cells (macrophages) in the liver, spleen, and bone marrow. Treatment failures due to emerging drug resistance are a significant concern. Using a combination of genetic and biochemical approaches, we have confirmed the mechanisms by which these parasites become less sensitive to treatment with amphotericin B. In addition, we have identified a novel mechanism involving loss of a key enzyme (cytochrome P450 reductase 1) in the biosynthetic pathway to ergosterol, an important lipid component of the parasite's plasma membrane. These studies increase our fundamental understanding of this important metabolic pathway and provide information that may be exploited to develop novel therapeutic strategies to combat this killer disease. [ABSTRACT FROM AUTHOR]
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- 2024
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9. Phased chromosome-scale genome assembly of an asexual, allopolyploid root-knot nematode reveals complex subgenomic structure.
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Winter, Michael R., Taranto, Adam P., Yimer, Henok Zemene, Coomer Blundell, Alison, Siddique, Shahid, Williamson, Valerie M., and Lunt, David H.
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ROOT-knot nematodes ,ROOT-knot ,PLANT nematodes ,JAVANESE root-knot nematode ,AGRICULTURE ,GENOMES - Abstract
We present the chromosome-scale genome assembly of the allopolyploid root-knot nematode Meloidogyne javanica. We show that the M. javanica genome is predominantly allotetraploid, comprising two subgenomes, A and B, that most likely originated from hybridisation of two ancestral parental species. The assembly was annotated using full-length non-chimeric transcripts, comparison to reference databases, and ab initio prediction techniques, and the subgenomes were phased using ancestral k-mer spectral analysis. Subgenome B appears to show fission of chromosomal contigs, and while there is substantial synteny between subgenomes, we also identified regions lacking synteny that may have diverged in the ancestral genomes prior to or following hybridisation. This annotated and phased genome assembly forms a significant resource for understanding the origins and genetics of these globally important plant pathogens. Author summary: Root-knot nematodes represent one of the most significant crop parasites globally. Despite their agricultural importance, only limited genomic resources have been published to date, leaving a gap in the understanding of genetic mechanisms driving genome evolution and crop virulence. Here, we have used modern genomic and bioinformatic approaches to create a chromosome-scale reference assembly to investigate the origins and genomic composition of the root-knot nematode species Meloidogyne javanica. This species has an allopolyploid genome, reproduces by ameiotic parthenogenesis and is among the most damaging plant parasitic nematodes with a large and expanding plant host range. Utilising modern long-read DNA sequencing and bioinformatics approaches, we successfully phased the assembly into its constituent subgenomes, a first for this agriculturally important clade. While we find the genomic landscape is mostly syntenic between subgenomes, we identified regions of minimal similarity, and highlight structural divergence between subgenomes. [ABSTRACT FROM AUTHOR]
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- 2024
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10. In the hunt for genomic markers of metabolic resistance to pyrethroids in the mosquito Aedes aegypti: An integrated next-generation sequencing approach.
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Faucon, Frederic, Gaude, Thierry, Dusfour, Isabelle, Navratil, Vincent, Corbel, Vincent, Juntarajumnong, Waraporn, Girod, Romain, Poupardin, Rodolphe, Boyer, Frederic, Reynaud, Stephane, and David, Jean-Philippe
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AEDES aegypti ,ARBOVIRUS diseases ,PYRETHROIDS ,GENOMICS ,GENE expression - Abstract
Background: The capacity of Aedes mosquitoes to resist chemical insecticides threatens the control of major arbovirus diseases worldwide. Until alternative control tools are widely deployed, monitoring insecticide resistance levels and identifying resistance mechanisms in field mosquito populations is crucial for implementing appropriate management strategies. Metabolic resistance to pyrethroids is common in Aedes aegypti but the monitoring of the dynamics of resistant alleles is impeded by the lack of robust genomic markers. Methodology/Principal findings: In an attempt to identify the genomic bases of metabolic resistance to deltamethrin, multiple resistant and susceptible populations originating from various continents were compared using both RNA-seq and a targeted DNA-seq approach focused on the upstream ATG regions of detoxification genes. Multiple detoxification enzymes were over transcribed in resistant populations, frequently associated with an increase in their gene copy number. Targeted sequencing identified potential promoter variations associated with their over transcription. Non-synonymous variations affecting detoxification enzymes were also identified in resistant populations. Conclusion /Significance: This study not only confirmed the role of gene copy number variations as a frequent cause of the over expression of detoxification enzymes associated with insecticide resistance in Aedes aegypti but also identified novel genomic resistance markers potentially associated with their cis-regulation and modifications of their protein structure conformation. As for gene transcription data, polymorphism patterns were frequently conserved within regions but differed among continents confirming the selection of different resistance factors worldwide. Overall, this study paves the way of the identification of a comprehensive set of genomic markers for monitoring the spatio-temporal dynamics of the variety of insecticide resistance mechanisms in Aedes aegypti. [ABSTRACT FROM AUTHOR]
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- 2017
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11. The ace-1 Locus Is Amplified in All Resistant Anopheles gambiae Mosquitoes: Fitness Consequences of Homogeneous and Heterogeneous Duplications.
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Assogba, Benoît S., Milesi, Pascal, Djogbénou, Luc S., Berthomieu, Arnaud, Makoundou, Patrick, Baba-Moussa, Lamine S., Fiston-Lavier, Anna-Sophie, Belkhir, Khalid, Labbé, Pierrick, and Weill, Mylène
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ANOPHELES gambiae ,MOSQUITOES ,GENES ,INSECT populations ,INSECT phenology - Abstract
Gene copy-number variations are widespread in natural populations, but investigating their phenotypic consequences requires contemporary duplications under selection. Such duplications have been found at the ace-1 locus (encoding the organophosphate and carbamate insecticides’ target) in the mosquito Anopheles gambiae (the major malaria vector); recent studies have revealed their intriguing complexity, consistent with the involvement of various numbers and types (susceptible or resistant to insecticide) of copies. We used an integrative approach, from genome to phenotype level, to investigate the influence of duplication architecture and gene-dosage on mosquito fitness. We found that both heterogeneous (i.e., one susceptible and one resistant ace-1 copy) and homogeneous (i.e., identical resistant copies) duplications segregated in field populations. The number of copies in homogeneous duplications was variable and positively correlated with acetylcholinesterase activity and resistance level. Determining the genomic structure of the duplicated region revealed that, in both types of duplication, ace-1 and 11 other genes formed tandem 203kb amplicons. We developed a diagnostic test for duplications, which showed that ace-1 was amplified in all 173 resistant mosquitoes analyzed (field-collected in several African countries), in heterogeneous or homogeneous duplications. Each type was associated with different fitness trade-offs: heterogeneous duplications conferred an intermediate phenotype (lower resistance and fitness costs), whereas homogeneous duplications tended to increase both resistance and fitness cost, in a complex manner. The type of duplication selected seemed thus to depend on the intensity and distribution of selection pressures. This versatility of trade-offs available through gene duplication highlights the importance of large mutation events in adaptation to environmental variation. This impressive adaptability could have a major impact on vector control in Africa. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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12. PD-L1 Status in Refractory Lymphomas.
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Vranic, Semir, Ghosh, Nilanjan, Kimbrough, Jeffery, Bilalovic, Nurija, Bender, Ryan, Arguello, David, Veloso, Yvonne, Dizdarevic, Aida, and Gatalica, Zoran
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HODGKIN'S disease treatment ,HODGKIN'S disease ,GENE expression ,B cells ,BIOLOGICAL assay ,DNA copy number variations ,PATIENTS - Abstract
Targeted immunotherapy based on PD-1/PD-L1 suppression has revolutionized the treatment of various solid tumors. A remarkable improvement has also been observed in the treatment of patients with refractory/relapsing classical Hodgkin lymphoma (cHL). We investigated PD-L1 status in a variety of treatment resistant lymphomas. Tumor samples from 78 patients with therapy resistant lymphomas were immunohistochemically (IHC) investigated for the expression of PD-L1 using two antibody clones (SP142 and SP263, Ventana). Thirteen PD-L1+ cases were further analyzed for gene copy number variations (CNV) by NGS and for PD-L1/JAK2/PD-L2 co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity (≥5% positive cancer cells, IHC) was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently negative (3/3). Co-amplification/CNVs of PD-L1/JAK2/PD-L2 were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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13. FISHtrees 3.0: Tumor Phylogenetics Using a Ploidy Probe.
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Gertz, E. Michael, Chowdhury, Salim Akhter, Lee, Woei-Jyh, Wangsa, Darawalee, Heselmeyer-Haddad, Kerstin, Ried, Thomas, Schwartz, Russell, and Schäffer, Alejandro A.
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FLUORESCENCE in situ hybridization ,PHYLOGENY ,CANCER invasiveness ,NUCLEOTIDE sequencing ,DNA copy number variations ,POLYPLOIDY ,CELL populations - Abstract
Advances in fluorescence in situ hybridization (FISH) make it feasible to detect multiple copy-number changes in hundreds of cells of solid tumors. Studies using FISH, sequencing, and other technologies have revealed substantial intra-tumor heterogeneity. The evolution of subclones in tumors may be modeled by phylogenies. Tumors often harbor aneuploid or polyploid cell populations. Using a FISH probe to estimate changes in ploidy can guide the creation of trees that model changes in ploidy and individual gene copy-number variations. We present FISHtrees 3.0, which implements a ploidy-based tree building method based on mixed integer linear programming (MILP). The ploidy-based modeling in FISHtrees includes a new formulation of the problem of merging trees for changes of a single gene into trees modeling changes in multiple genes and the ploidy. When multiple samples are collected from each patient, varying over time or tumor regions, it is useful to evaluate similarities in tumor progression among the samples. Therefore, we further implemented in FISHtrees 3.0 a new method to build consensus graphs for multiple samples. We validate FISHtrees 3.0 on a simulated data and on FISH data from paired cases of cervical primary and metastatic tumors and on paired breast ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Tests on simulated data show improved accuracy of the ploidy-based approach relative to prior ploidyless methods. Tests on real data further demonstrate novel insights these methods offer into tumor progression processes. Trees for DCIS samples are significantly less complex than trees for paired IDC samples. Consensus graphs show substantial divergence among most paired samples from both sets. Low consensus between DCIS and IDC trees may help explain the difficulty in finding biomarkers that predict which DCIS cases are at most risk to progress to IDC. The FISHtrees software is available at . [ABSTRACT FROM AUTHOR]
- Published
- 2016
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14. The genome formula of a multipartite virus is regulated both at the individual segment and the segment group levels.
- Author
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Bonnamy, Mélia, Brousse, Andy, Pirolles, Elodie, Michalakis, Yannis, and Blanc, Stéphane
- Subjects
FAVA bean ,VIRAL genomes ,GENOMES ,SINGLE-stranded DNA ,GENE expression - Abstract
Differential accumulation of the distinct genome segments is a common feature of viruses with segmented genomes. The reproducible and specific pattern of genome segment accumulation within the host is referred to as the "genome formula". There is speculation and some experimental support for a functional role of the genome formula by modulating gene expression through copy number variations. However, the mechanisms of genome formula regulation have not yet been identified. In this study, we investigated whether the genome formula of the octopartite nanovirus faba bean necrotic stunt virus (FBNSV) is regulated by processes acting at the individual segment vs. viral population levels. We used a leaf infiltration system to show that the two most accumulated genome segments of the FBNSV possess a greater intrinsic accumulation capacity in Vicia faba tissues than the other segments. Nevertheless, processes acting at the individual segment level are insufficient to generate the genome formula, suggesting the involvement of additional mechanisms acting at the supra-segment level. Indeed, the absence of segments with important functions during systemic infection strongly modifies the relative frequency of the others, indicating that the genome formula is a property of the segment group. Together, these results demonstrate that the FBNSV genome formula is shaped by a complex process acting at both the individual segment and the segment group levels. Author summary: Segmented and multipartite viruses have their genomic information carried by several molecules, which allows for the unequal accumulation of the different genome segments in infected tissues. The reproducible and specific pattern of genome segment accumulation within the host is referred to as the "genome formula". The genome formula is host-dependent and believed to modulate gene expression through copy number variations upon host switches. The mechanisms leading to the genome formula remain unknown. Here, we determined that the genome formula of an octopartite single-stranded DNA nanovirus is shaped by processes acting both at the level of individual segments, some having higher accumulation rates, and at the level of the group of segments, the omission of some non-essential segments affecting the relative accumulation of others. Our study provides insights into the level at which genome formula regulation operates, giving a starting point for future studies aiming at elucidating the mechanisms governing genome formula in multipartite and segmented viruses. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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15. Molecular Integrative Clustering of Asian Gastric Cell Lines Revealed Two Distinct Chemosensitivity Clusters.
- Author
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Choong, Meng Ling, Tan, Shan Ho, Tan, Tuan Zea, Manesh, Sravanthy, Ngo, Anna, Yong, Jacklyn W. Y., Yang, Henry He, and Lee, May Ann
- Subjects
CELL lines ,GASTROINTESTINAL system ,CELL culture ,GENES ,GENOMICS ,GASTRIC diseases - Abstract
Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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16. Genetic Analysis of Leishmania donovani Tropism Using a Naturally Attenuated Cutaneous Strain.
- Author
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Zhang, Wen Wei, Ramasamy, Gowthaman, McCall, Laura-Isobel, Haydock, Andrew, Ranasinghe, Shalindra, Abeygunasekara, Priyanka, Sirimanna, Ganga, Wickremasinghe, Renu, Myler, Peter, and Matlashewski, Greg
- Subjects
LEISHMANIA donovani ,VISCERAL leishmaniasis ,PSEUDOGENES ,LABORATORY mice ,CUTANEOUS leishmaniasis ,VIRAL tropism - Abstract
A central question in Leishmania research is why most species cause cutaneous infections but others cause fatal visceral disease. Interestingly, L. donovani causes both visceral and cutaneous leishmaniasis in Sri Lanka. L. donovani clinical isolates were therefore obtained from cutaneous leishmaniasis (CL-SL) and visceral leishmaniasis (VL-SL) patients from Sri Lanka. The CL-SL isolate was severely attenuated compared to the VL-SL isolate for survival in visceral organs in BALB/c mice. Genomic and transcriptomic analysis argue that gene deletions or pseudogenes specific to CL-SL are not responsible for the difference in disease tropism and that single nucleotide polymorphisms (SNPs) and/or gene copy number variations play a major role in altered pathology. This is illustrated through the observations within showing that a decreased copy number of the A2 gene family and a mutation in the ras-like RagC GTPase enzyme in the mTOR pathway contribute to the attenuation of the CL-SL strain in visceral infection. Overall, this research provides a unique perspective on genetic differences associated with diverse pathologies caused by Leishmania infection. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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17. Integrative Analysis of Transcriptional Regulatory Network and Copy Number Variation in Intrahepatic Cholangiocarcinoma.
- Author
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Li, Ling, Lian, Baofeng, Li, Chao, Li, Wei, Li, Jing, Zhang, Yuannv, He, Xianghuo, Li, Yixue, and Xie, Lu
- Subjects
CHOLANGIOCARCINOMA ,GENE regulatory networks ,DNA copy number variations ,LIVER cancer ,CELLULAR signal transduction ,GENETICS ,PROGNOSIS - Abstract
Background: Transcriptional regulatory network (TRN) is used to study conditional regulatory relationships between transcriptional factors and genes. However few studies have tried to integrate genomic variation information such as copy number variation (CNV) with TRN to find causal disturbances in a network. Intrahepatic cholangiocarcinoma (ICC) is the second most common hepatic carcinoma with high malignancy and poor prognosis. Research about ICC is relatively limited comparing to hepatocellular carcinoma, and there are no approved gene therapeutic targets yet. Method: We first constructed TRN of ICC (ICC-TRN) using forward-and-reverse combined engineering method, and then integrated copy number variation information with ICC-TRN to select CNV-related modules and constructed CNV-ICC-TRN. We also integrated CNV-ICC-TRN with KEGG signaling pathways to investigate how CNV genes disturb signaling pathways. At last, unsupervised clustering method was applied to classify samples into distinct classes. Result: We obtained CNV-ICC-TRN containing 33 modules which were enriched in ICC-related signaling pathways. Integrated analysis of the regulatory network and signaling pathways illustrated that CNV might interrupt signaling through locating on either genomic sites of nodes or regulators of nodes in a signaling pathway. In the end, expression profiles of nodes in CNV-ICC-TRN were used to cluster the ICC patients into two robust groups with distinct biological function features. Conclusion: Our work represents a primary effort to construct TRN in ICC, also a primary effort to try to identify key transcriptional modules based on their involvement of genetic variations shown by gene copy number variations (CNV). This kind of approach may bring the traditional studies of TRN based only on expression data one step further to genetic disturbance. Such kind of approach can easily be extended to other disease samples with appropriate data. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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18. Variations in Stress Sensitivity and Genomic Expression in Diverse S. cerevisiae Isolates.
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Kvitek, Daniel J., Will, Jessica L., and Gasch, Audrey P.
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BIOLOGICAL variation ,GENE expression ,SACCHAROMYCES ,PHENOTYPIC plasticity ,PHYSIOLOGICAL stress ,YEAST fungi ,GENOMICS - Abstract
Interactions between an organism and its environment can significantly influence phenotypic evolution. A first step toward understanding this process is to characterize phenotypic diversity within and between populations. We explored the phenotypic variation in stress sensitivity and genomic expression in a large panel of Saccharomyces strains collected from diverse environments. We measured the sensitivity of 52 strains to 14 environmental conditions, compared genomic expression in 18 strains, and identified gene copy-number variations in six of these isolates. Our results demonstrate a large degree of phenotypic variation in stress sensitivity and gene expression. Analysis of these datasets reveals relationships between strains from similar niches, suggests common and unique features of yeast habitats, and implicates genes whose variable expression is linked to stress resistance. Using a simple metric to suggest cases of selection, we found that strains collected from oak exudates are phenotypically more similar than expected based on their genetic diversity, while sake and vineyard isolates display more diverse phenotypes than expected under a neutral model. We also show that the laboratory strain S288c is phenotypically distinct from all of the other strains studied here, in terms of stress sensitivity, gene expression, Ty copy number, mitochondrial content, and gene-dosage control. These results highlight the value of understanding the genetic basis of phenotypic variation and raise caution about using laboratory strains for comparative genomics. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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19. Cheating leads to the evolution of multipartite viruses.
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Leeks, Asher, Young, Penny Grace, Turner, Paul Eugene, Wild, Geoff, and West, Stuart Andrew
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MIXED infections ,GENOMES ,GENES ,HYPOTHESIS - Abstract
In multipartite viruses, the genome is split into multiple segments, each of which is transmitted via a separate capsid. The existence of multipartite viruses poses a problem, because replication is only possible when all segments are present within the same host. Given this clear cost, why is multipartitism so common in viruses? Most previous hypotheses try to explain how multipartitism could provide an advantage. In so doing, they require scenarios that are unrealistic and that cannot explain viruses with more than 2 multipartite segments. We show theoretically that selection for cheats, which avoid producing a shared gene product, but still benefit from gene products produced by other genomes, can drive the evolution of both multipartite and segmented viruses. We find that multipartitism can evolve via cheating under realistic conditions and does not require unreasonably high coinfection rates or any group-level benefit. Furthermore, the cheating hypothesis is consistent with empirical patterns of cheating and multipartitism across viruses. More broadly, our results show how evolutionary conflict can drive new patterns of genome organisation in viruses and elsewhere. In multipartite viruses, the genome is split into multiple segments, each of which is transmitted via a separate capsid. This seems costly, so why is this form of genome organization so widespread? This theoretical study shows that selection for cheats can drive the evolution of multipartite viruses under realistic conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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20. Neural networks enable efficient and accurate simulation-based inference of evolutionary parameters from adaptation dynamics.
- Author
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Avecilla, Grace, Chuong, Julie N., Li, Fangfei, Sherlock, Gavin, Gresham, David, and Ram, Yoav
- Subjects
DNA copy number variations ,ARTIFICIAL neural networks ,EVOLUTIONARY models ,CELL division - Abstract
The rate of adaptive evolution depends on the rate at which beneficial mutations are introduced into a population and the fitness effects of those mutations. The rate of beneficial mutations and their expected fitness effects is often difficult to empirically quantify. As these 2 parameters determine the pace of evolutionary change in a population, the dynamics of adaptive evolution may enable inference of their values. Copy number variants (CNVs) are a pervasive source of heritable variation that can facilitate rapid adaptive evolution. Previously, we developed a locus-specific fluorescent CNV reporter to quantify CNV dynamics in evolving populations maintained in nutrient-limiting conditions using chemostats. Here, we use CNV adaptation dynamics to estimate the rate at which beneficial CNVs are introduced through de novo mutation and their fitness effects using simulation-based likelihood–free inference approaches. We tested the suitability of 2 evolutionary models: a standard Wright–Fisher model and a chemostat model. We evaluated 2 likelihood-free inference algorithms: the well-established Approximate Bayesian Computation with Sequential Monte Carlo (ABC-SMC) algorithm, and the recently developed Neural Posterior Estimation (NPE) algorithm, which applies an artificial neural network to directly estimate the posterior distribution. By systematically evaluating the suitability of different inference methods and models, we show that NPE has several advantages over ABC-SMC and that a Wright–Fisher evolutionary model suffices in most cases. Using our validated inference framework, we estimate the CNV formation rate at the GAP1 locus in the yeast Saccharomyces cerevisiae to be 10
−4.7 to 10−4 CNVs per cell division and a fitness coefficient of 0.04 to 0.1 per generation for GAP1 CNVs in glutamine-limited chemostats. We experimentally validated our inference-based estimates using 2 distinct experimental methods—barcode lineage tracking and pairwise fitness assays—which provide independent confirmation of the accuracy of our approach. Our results are consistent with a beneficial CNV supply rate that is 10-fold greater than the estimated rates of beneficial single-nucleotide mutations, explaining the outsized importance of CNVs in rapid adaptive evolution. More generally, our study demonstrates the utility of novel neural network–based likelihood–free inference methods for inferring the rates and effects of evolutionary processes from empirical data with possible applications ranging from tumor to viral evolution. This study shows that simulation-based inference of evolutionary dynamics using neural networks can yield parameter values for fitness and mutation rate that are difficult to determine experimentally, including those of copy number variants (CNVs) during experimental adaptive evolution of yeast. [ABSTRACT FROM AUTHOR]- Published
- 2022
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21. Experimental evolution links post-transcriptional regulation to Leishmania fitness gain.
- Author
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Piel, Laura, Rajan, K. Shanmugha, Bussotti, Giovanni, Varet, Hugo, Legendre, Rachel, Proux, Caroline, Douché, Thibaut, Giai-Gianetto, Quentin, Chaze, Thibault, Cokelaer, Thomas, Vojtkova, Barbora, Gordon-Bar, Nadav, Doniger, Tirza, Cohen-Chalamish, Smadar, Rengaraj, Praveenkumar, Besse, Céline, Boland, Anne, Sadlova, Jovana, Deleuze, Jean-François, and Matondo, Mariette
- Subjects
LEISHMANIASIS ,LEISHMANIA ,VISCERAL leishmaniasis ,RNA regulation ,LEISHMANIA donovani ,PHENOTYPIC plasticity - Abstract
The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings. Author summary: Genome instability plays a central yet poorly understood role in human disease. Gene amplifications and deletions drive cancer development, microbial infection and therapeutic failure. The molecular mechanisms that harness the deleterious effects of genome instability to generate beneficial phenotypes in pathogenic systems are unknown. Here we study this important open question in the protozoan parasite Leishmania that causes devastating human diseases termed leishmaniases. Leishmania parasites lack transcriptional control and instead exploit genome instability to adapt to their host environment. Analyzing in vitro adaptation of hamster-derived parasites via gene copy number (genomic level) and gene expression changes (transcriptomic and proteomic levels), we show that these parasites likely exploit small nucleolar RNAs (snoRNAs) to mitigate toxic effects of genome instability by post-transcriptional regulation and the establishment of modified ribosomes. Our findings propose non-coding RNAs as potential novel biomarkers with diagnostic and prognostic value that may be linked to changes in parasite tissue tropism or drug susceptibility. This novel insight into Leishmania adaptation will be likely applicable to other fast evolving eukaryotic systems with unstable genomes, such as fungi or cancer cells. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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- View/download PDF
22. High genome plasticity and frequent genetic exchange in Leishmania tropica isolates from Afghanistan, Iran and Syria.
- Author
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Glans, Hedvig, Lind Karlberg, Maria, Advani, Reza, Bradley, Maria, Alm, Erik, Andersson, Björn, and Downing, Tim
- Subjects
LEISHMANIA ,CUTANEOUS leishmaniasis ,GENETIC variation ,GENOMES ,GENETIC recombination ,TREATMENT failure - Abstract
Background: The kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica's digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss. Methodology/principal findings: The genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007–2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units. Conclusion/significance: These large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East. Author summary: Cutaneous leishmaniasis is mainly caused by Leishmania tropica in the Middle East, where it is known for treatment failure and a need for prolonged and/or multiple treatments. Several factors affect the clinical presentation and treatment outcome, such as host genetic variability and specific immune response, as well as environmental factors and the vector species. Little is known about the parasite genome and its influence on treatment response. By analysing the genome of 22 isolates of L. tropica, we have revealed extensive genomic variation and a complex population structure with evidence of genetic exchange within and among the isolates, indicating a possible presence of sexual or parasexual mechanisms. Understanding the Leishmania genome better may improve future treatment and better understanding of treatment failure and relapse. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
23. Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana.
- Author
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Poláková, Ester, Albanaz, Amanda T. S., Zakharova, Alexandra, Novozhilova, Tatiana S., Gerasimov, Evgeny S., and Yurchenko, Vyacheslav
- Subjects
LEISHMANIA mexicana ,TELOMERES ,NEGLECTED diseases ,TRYPANOSOMA brucei ,NUCLEOTIDE sequencing - Abstract
Background: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. Methodology/Principal findings: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. Conclusion/Significance: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity. Author summary: Parasites of the genus Leishmania cause leishmaniasis, a neglected tropical disease. Telomeric and sub-telomeric regions of these flagellates are often associated with virulence. That is why studying telomere maintenance is important. Here we demonstrate that deletion of one of the proteins, implicated in telomere maintenance, resulted in elongated telomeres. This is unexpected, because deletion of the same gene in another model organism, Trypanosoma brucei, has an opposite effect. Previous comparative genomic studies suggested that the protein under study might be also involved in genome stability. Nevertheless, we did not detect any trace of genomic instability in L. mexicana after 100 passages. We concluded that Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
24. Decreased glutamate transport in acivicin resistant Leishmania tarentolae.
- Author
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Roy, Gaétan, Bhattacharya, Arijit, Leprohon, Philippe, and Ouellette, Marc
- Subjects
GLUTAMIC acid ,GLUTAMATE transporters ,GENETIC code ,GENETIC overexpression ,LEISHMANIA ,AMINO acids ,LEISHMANIA mexicana - Abstract
Studies of drug resistance in the protozoan parasites of the genus Leishmania have been helpful in revealing biochemical pathways as potential drug targets. The chlorinated glutamine analogue acivicin has shown good activity against Leishmania cells and was shown to target several enzymes containing amidotransferase domains. We selected a Leishmania tarentolae clone for acivicin resistance. The genome of this resistant strain was sequenced and the gene coding for the amidotransferase domain-containing GMP synthase was found to be amplified. Episomal expression of this gene in wild-type L. tarentolae revealed a modest role in acivicin resistance. The most prominent defect observed in the resistant mutant was reduced uptake of glutamate, and through competition experiments we determined that glutamate and acivicin, but not glutamine, share the same transporter. Several amino acid transporters (AATs) were either deleted or mutated in the resistant cells. Some contributed to the acivicin resistance phenotype although none corresponded to the main glutamate transporter. Through sequence analysis one AAT on chromosome 22 corresponded to the main glutamate transporter. Episomal expression of the gene coding for this transporter in the resistant mutant restored glutamate transport and acivicin susceptibility. Its genetic knockout led to reduced glutamate transport and acivicin resistance. We propose that acivicin binds covalently to this transporter and as such leads to decreased transport of glutamate and acivicin thus leading to acivicin resistance. Author summary: Studies of drug resistance in the protozoan parasites of the genus Leishmania have been helpful in revealing biochemical pathways as potential drug targets. Here we report on the characterization at the genomics and metabolomics levels of a L. tarentolae strain made resistant to acivicin, an analogue of glutamine with activity against this parasite. We found that resistance to acivicin is accompanied by a reduced uptake and intracellular levels of glutamate and that both are expected to share the same transporter. Through gene overexpression and disruption studies we identified the main amino acid transporter responsible for glutamate uptake. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
25. Combined gene deletion of dihydrofolate reductase-thymidylate synthase and pteridine reductase in Leishmania infantum.
- Author
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Bhattacharya, Arijit, Leprohon, Philippe, and Ouellette, Marc
- Subjects
LEISHMANIA infantum ,DELETION mutation ,THYMIDYLATE synthase ,PTERIDINES ,NUCLEOSIDE transport proteins ,DRUG metabolism - Abstract
Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously. Author summary: The protozoan parasite Leishmania is auxotroph for folate and unconjugated pterins and salvages both from the mammalian host. Two enzymes of the folate metabolism pathway, namely the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the pteridine reductase 1 (PTR1), are being evaluated for drug discovery and repurposing of existing anti-metabolites. Despite their apparent potential, development of DHFR-TS and PTR1 targeted chemotherapy against Leishmania is still awaiting. Here we revisited folate metabolism at the genomic level and report on the identification of known resistance genes alongside some new ones. Through gene disruption studies we found that L. infantum DHFR-TS null mutants are thymidine auxotroph and that these can be rescued by the bacterial flavin dependent thymidylate synthase ThyX. We also found that PTR1 is essential in the absence of a functional DHFR-TS even in the presence of ThyX or thymidine supplementation, indicating the essential role of reduced pterins or folate beyond thymidine synthesis. This study indicates that simultaneous targeting of DHFR-TS and PTR1 will be required for the development of anti-folate chemotherapy against Leishmania. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
26. Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance.
- Author
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Turkiewicz, Anna, Manko, Emilia, Sutherland, Colin J., Diez Benavente, Ernest, Campino, Susana, and Clark, Taane G.
- Subjects
TETRAHYDROFOLATE dehydrogenase ,PLASMODIUM falciparum ,DRUG resistance ,BACTERIAL diseases ,PLASMODIUM - Abstract
Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms. Author summary: Malaria causes approximately 435,000 deaths per year, concentrated in sub-Saharan Africa and among children under the age of five years. Global efforts to control and eliminate malaria are hampered by the emergence of Plasmodium falciparum malaria parasites resistant to currently available antimalarial drugs. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria and, although replaced by artemisinin combinations, is still used for the prevention of malarial disease in vulnerable groups (e.g. pregnant women). SP resistance is caused by mutations in the P. falciparum parasite genes pfdhfr and pfdhps, but recently novel structural variants in and around the pfgch1 gene have been described. By analysing genome sequence data of 4,134 P. falciparum across 29 malaria endemic countries, we establish there are at least ten different pfgch1 structural variants, existing in the presence of pfdhfr and pfdhps mutations, and occur increasingly and at high frequency in some Southeast Asian and African populations. These pfgch1 structural variants may enhance the survival of those parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of SP for prevention of malaria in vulnerable groups. Therefore it is important that they are monitored in molecular surveillance studies. Our work will assist epidemiological studies, laboratory and surveillance activities looking at the diversity and role of pfgch1 structural variants and their prevalence across malaria endemic regions, especially in countries using SP regimens. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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- View/download PDF
27. Homogeneity among glyphosate-resistant Amaranthus palmeri in geographically distant locations.
- Author
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Molin, William T., Patterson, Eric L., and Saski, Christopher A.
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AMARANTHUS palmeri ,GLYPHOSATE ,EXTRACHROMOSOMAL DNA ,GENE amplification ,CIRCULAR DNA ,SHOTGUN sequencing ,HOMOGENEITY - Abstract
Since the initial report of glyphosate-resistant (GR) Amaranthus palmeri S. Watson in 2006, resistant populations have been reported in 28 states. The mechanism of resistance is amplification of a 399-kb extrachromosomal circular DNA, called the EPSPS replicon, and is unique to glyphosate-resistant plants. The replicon contains a single copy of the 10-kb 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene which causes the concomitant increased expression of EPSP synthase, the target enzyme of glyphosate. It is not known whether the resistance by this amplification mechanism evolved once and then spread across the country or evolved independently in several locations. To compare genomic representation and variation across the EPSPS replicon, whole genome shotgun sequencing (WGS) and mapping of sequences from both GR and susceptible (GS) biotypes to the replicon consensus sequence was performed. Sampling of GR biotypes from AZ, KS, GA, MD and DE and GS biotypes from AZ, KS and GA revealed complete contiguity and deep representation with sequences from GR plants, but lack of homogeneity and contiguity with breaks in coverage were observed with sequences from GS biotypes. The high sequence conservation among GR biotypes with very few polymorphisms which were widely distributed across the USA further supports the hypothesis that glyphosate resistance most likely originated from a single population. We show that the replicon from different populations was unique to GR plants and had similar levels of amplification. [ABSTRACT FROM AUTHOR]
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- 2020
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28. KAT6A amplifications are associated with shorter progression-free survival and overall survival in patients with endometrial serous carcinoma.
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Saglam, Ozlen, Tang, Zhenya, Tang, Guilin, Medeiros, L. Jeffrey, and Toruner, Gokce A.
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PROGRESSION-free survival ,CARCINOMA ,GENE expression ,ENDOMETRIUM ,SOMATIC mutation ,DISEASE progression ,HUMAN embryo transfer ,ENDOMETRIOSIS - Abstract
Somatic copy number alterations (CNA) are common in endometrial serous carcinoma (ESC). We used the Tumor Cancer Genome Atlas Pan Cancer dataset (TCGA Pan Can) to explore the impact of somatic CNA and gene expression levels (mRNA) of cancer-related genes in ESC. Results were correlated with clinico-pathologic parameters such as age of onset, disease stage, progression-free survival (PFS) and overall survival (OS) (n = 108). 1,449 genes with recurrent somatic CNA were identified, observed in 10% or more tumor samples. Somatic CNA and mRNA expression levels were highly correlated (r> = 0.6) for 383 genes. Among these, 45 genes were classified in the Tier 1 category of Cancer Genome Census-Catalogue of Somatic Mutations in Cancer. Eighteen of 45 Tier 1 genes had highly correlated somatic CNA and mRNA expression levels including ARNT, PIK3CA, TBLXR1, ASXL1, EIF4A2, HOOK3, IKBKB, KAT6A, TCEA1, KAT6B, ERBB2, BRD4, KEAP1, PRKACA, DNM2, SMARCA4, AKT2, SS18L1. Our results are in agreement with previously reported somatic CNA for ERBB2, BRD4 and PIK3C in ESC. In addition, AKT2 (p = 0.002) and KAT6A (p = 0.015) amplifications were more frequent in tumor samples from younger patients (<60), and CEBPA (p = 0.028) and MYC (p = 0.023) amplifications were more common with advanced (stage III and IV) disease stage. Patients with tumors carrying KAT6A and MYC amplifications had shorter PFS and OS. The hazard ratio (HR) of KAT6A was 2.82 [95 CI 1.12–7.07] for PFS and 3.87 [95 CI 1.28–11.68] for OS. The HR of MYC was 2.25 [95 CI 1.05–4.81] and 2.62[95 CI 1.07–6.41] for PFS and OS, respectively. [ABSTRACT FROM AUTHOR]
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- 2020
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29. In-depth quantitative proteomics uncovers specie-specific metabolic programs in Leishmania (Viannia) species.
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Pinho, Nathalia, Wiśniewski, Jacek R., Dias-Lopes, Geovane, Saboia-Vahia, Leonardo, Bombaça, Ana Cristina Souza, Mesquita-Rodrigues, Camila, Menna-Barreto, Rubem, Cupolillo, Elisa, de Jesus, Jose Batista, Padrón, Gabriel, and Cuervo, Patricia
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LEISHMANIA mexicana ,PROTEOMICS ,LEISHMANIA ,MEMBRANE proteins ,PRINCIPAL components analysis ,ENERGY metabolism - Abstract
Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O
2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696. Author summary: Leishmania braziliensis, L. panamensis, and L. guyanensis are responsible for most of the cases of tegumentary leishmaniasis (TL) in the Americas. These species are associated with a variety of clinical manifestations of TL ranging from self-healing localized cutaneous lesions to disseminated and mucocutaneous presentations that may result in severe oropharyngeal mutilation. Here, we report a comprehensive quantitative comparison of the proteome of those species. Assessment of absolute titers of ~7000 proteins revealed a very clear differentiation among them. Significant differences in energy metabolism, membrane proteins, transporters, and lipids are contributing for species-specific traits and provide rich substrate for exploring new molecules for diagnosing purposes. [ABSTRACT FROM AUTHOR]- Published
- 2020
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30. Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication.
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Damasceno, Jeziel D., Reis-Cunha, João, Crouch, Kathryn, Beraldi, Dario, Lapsley, Craig, Tosi, Luiz R. O., Bartholomeu, Daniella, and McCulloch, Richard
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LEISHMANIA major ,HOMOLOGOUS recombination ,DNA synthesis ,DNA replication ,GENE knockout ,DNA damage ,DNA repair - Abstract
Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote. Author summary: Homologous recombination plays a key role in genome maintenance during cell division, but loss of factors directing the reaction has not been described as being lethal in any microbe. Here, we have used a genetic strategy to selectively induce loss, singly and doubly, of five genes in Leishmania that act in homologous recombination, revealing two things. First, loss of any gene related to RAD51, which catalyses homologous recombination, is not immediately lethal, but leads to increasing growth impairment and genome damage accumulation. Second, loss of RAD51 causes a pronounced change in the programme of Leishmania genome replication. Thus, we show that homologous recombination in Leishmania can be essential, in part due to an unanticipated role in genome transmission. [ABSTRACT FROM AUTHOR]
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- 2020
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31. Phylogenetic background and habitat drive the genetic diversification of Escherichia coli.
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Touchon, Marie, Perrin, Amandine, de Sousa, Jorge André Moura, Vangchhia, Belinda, Burn, Samantha, O'Brien, Claire L., Denamur, Erick, Gordon, David, and Rocha, Eduardo PC
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MOBILE genetic elements ,HORIZONTAL gene transfer ,DRUG resistance in bacteria ,FECAL contamination ,GENOME size ,COLIFORMS ,BACTERIAL adaptation ,ESCHERICHIA coli - Abstract
Escherichia coli is mostly a commensal of birds and mammals, including humans, where it can act as an opportunistic pathogen. It is also found in water and sediments. We investigated the phylogeny, genetic diversification, and habitat-association of 1,294 isolates representative of the phylogenetic diversity of more than 5,000 isolates from the Australian continent. Since many previous studies focused on clinical isolates, we investigated mostly other isolates originating from humans, poultry, wild animals and water. These strains represent the species genetic diversity and reveal widespread associations between phylogroups and isolation sources. The analysis of strains from the same sequence types revealed very rapid change of gene repertoires in the very early stages of divergence, driven by the acquisition of many different types of mobile genetic elements. These elements also lead to rapid variations in genome size, even if few of their genes rise to high frequency in the species. Variations in genome size are associated with phylogroup and isolation sources, but the latter determine the number of MGEs, a marker of recent transfer, suggesting that gene flow reinforces the association of certain genetic backgrounds with specific habitats. After a while, the divergence of gene repertoires becomes linear with phylogenetic distance, presumably reflecting the continuous turnover of mobile element and the occasional acquisition of adaptive genes. Surprisingly, the phylogroups with smallest genomes have the highest rates of gene repertoire diversification and fewer but more diverse mobile genetic elements. This suggests that smaller genomes are associated with higher, not lower, turnover of genetic information. Many of these genomes are from freshwater isolates and have peculiar traits, including a specific capsule, suggesting adaptation to this environment. Altogether, these data contribute to explain why epidemiological clones tend to emerge from specific phylogenetic groups in the presence of pervasive horizontal gene transfer across the species. Author summary: Previous large scale studies on the evolution of E. coli focused on clinical isolates emphasizing virulence and antibiotic resistance in medically important lineages. Yet, most E. coli strains are either human commensals or not associated with humans at all. Here, we analyzed a large collection of non-clinical isolates of the species to assess the mechanisms of gene repertoire diversification in the light of isolation sources and phylogeny. We show that gene repertoires evolve so rapidly by the high turnover of mobile genetic elements that epidemiologically indistinguishable strains can be phenotypically extremely heterogeneous, illustrating the velocity of bacterial adaptation and the importance of accounting for the information on the whole genome at the epidemiological scale. Phylogeny and habitat shape the genetic diversification of E. coli to similar extents. Surprisingly, freshwater strains seem specifically adapted to this environment, breaking the paradigm that E. coli environmental isolates are systematically fecal contaminations. As a consequence, the evolution of this species is also shaped by environmental habitats, and it may diversify by acquiring genes and mobile elements from environmental bacteria (and not just from gut bacteria). This may facilitate the acquisition of virulence factors and antibiotic resistance in the strains that become pathogenic. [ABSTRACT FROM AUTHOR]
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- 2020
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32. Genomic analysis of natural intra-specific hybrids among Ethiopian isolates of Leishmania donovani.
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Cotton, James A., Durrant, Caroline, Franssen, Susanne U., Gelanew, Tesfaye, Hailu, Asrat, Mateus, David, Sanders, Mandy J., Berriman, Matthew, Volf, Petr, Miles, Michael A., and Yeo, Matthew
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LEISHMANIA mexicana ,LEISHMANIA donovani ,VISCERAL leishmaniasis ,GENETIC recombination ,SAND flies ,PROTOZOAN diseases - Abstract
Parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis due to Leishmania donovani is endemic in Ethiopia where it has also been responsible for major epidemics. The presence of hybrid genotypes has been widely reported in surveys of natural populations, genetic variation reported in a number of Leishmania species, and the extant capacity for genetic exchange demonstrated in laboratory experiments. However, patterns of recombination and the evolutionary history of admixture that produced these hybrid populations remain unclear. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterized as hybrids by microsatellite and multi-locus sequencing. To date there is only one previous study on a natural population of Leishmania hybrids based on whole-genome sequences. We propose that these hybrids originate from recombination between two different lineages of Ethiopian L. donovani occurring in the same region. Patterns of inheritance are more complex than previously reported with multiple, apparently independent, origins from similar parents that include backcrossing with parental types. Analysis indicates that hybrids are representative of at least three different histories. Furthermore, isolates were highly polysomic at the level of chromosomes with differences between parasites recovered from a recrudescent infection from a previously treated individual. The results demonstrate that recombination is a significant feature of natural populations and contributes to the growing body of data that shows how recombination, and gene flow, shape natural populations of Leishmania. Author summary: Leishmaniasis is a spectrum of diseases caused by the protozoan parasite Leishmania. It is transmitted by sand fly insect vectors and is responsible for an enormous burden of human suffering. In this manuscript we examine Leishmania isolates from Ethiopia that cause the most serious form of the disease, namely visceral leishmaniasis, which is usually fatal without treatment. Historically the general view was that such parasites reproduce clonally, so that their progeny are genetically identical to the founding cells. This view has changed over time and it is increasingly clear that recombination between genetically different Leishmania parasites occurs. The implication is that new biological traits such as virulence, resistance to drug treatments or the ability to infect new species of sand fly could emerge. The frequency and underlying mechanism of such recombination in natural isolates is poorly understood. Here we perform a detailed whole genome analysis on a cohort of hybrid isolates from Ethiopia together with their potential parents to assess the genetic nature of hybrids in more detail. Results reveal a complex pattern of mating and inbreeding indicative of multiple mating events that has likely shaped the epidemiology of the disease agent. We also show that some hybrids have very different relative amounts of DNA (polysomy) the implications of which are discussed. Together the results contribute to a fuller understanding of the nature of genetic recombination in natural populations of Leishmania. [ABSTRACT FROM AUTHOR]
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- 2020
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33. A spontaneous complex structural variant in rcan-1 increases exploratory behavior and laboratory fitness of Caenorhabditis elegans.
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Zhao, Yuehui, Long, Lijiang, Wan, Jason, Biliya, Shweta, Brady, Shannon C., Lee, Daehan, Ojemakinde, Akinade, Andersen, Erik C., Vannberg, Fredrik O., Lu, Hang, and McGrath, Patrick T.
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CURIOSITY ,CAENORHABDITIS elegans ,ANIMAL behavior ,BEHAVIOR ,LABORATORY animals - Abstract
Over long evolutionary timescales, major changes to the copy number, function, and genomic organization of genes occur, however, our understanding of the individual mutational events responsible for these changes is lacking. In this report, we study the genetic basis of adaptation of two strains of C. elegans to laboratory food sources using competition experiments on a panel of 89 recombinant inbred lines (RIL). Unexpectedly, we identified a single RIL with higher relative fitness than either of the parental strains. This strain also displayed a novel behavioral phenotype, resulting in higher propensity to explore bacterial lawns. Using bulk-segregant analysis and short-read resequencing of this RIL, we mapped the change in exploration behavior to a spontaneous, complex rearrangement of the rcan-1 gene that occurred during construction of the RIL panel. We resolved this rearrangement into five unique tandem inversion/duplications using Oxford Nanopore long-read sequencing. rcan-1 encodes an ortholog to human RCAN1/DSCR1 calcipressin gene, which has been implicated as a causal gene for Down syndrome. The genomic rearrangement in rcan-1 creates two complete and two truncated versions of the rcan-1 coding region, with a variety of modified 5' and 3' non-coding regions. While most copy-number variations (CNVs) are thought to act by increasing expression of duplicated genes, these changes to rcan-1 ultimately result in the reduction of its whole-body expression due to changes in the upstream regions. By backcrossing this rearrangement into a common genetic background to create a near isogenic line (NIL), we demonstrate that both the competitive advantage and exploration behavioral changes are linked to this complex genetic variant. This NIL strain does not phenocopy a strain containing an rcan-1 loss-of-function allele, which suggests that the residual expression of rcan-1 is necessary for its fitness effects. Our results demonstrate how colonization of new environments, such as those encountered in the laboratory, can create evolutionary pressure to modify gene function. This evolutionary mismatch can be resolved by an unexpectedly complex genetic change that simultaneously duplicates and diversifies a gene into two uniquely regulated genes. Our work shows how complex rearrangements can act to modify gene expression in ways besides increased gene dosage. Author summary: Evolution acts on genetic variants that modify phenotypes that increase the likelihood of staying alive and passing on these genetic changes to subsequent generations (i.e. fitness). There is general interest in understanding the types of genetic variants that can increase fitness in specific environments. One route that fitness can be increased is through changes in behavior, such as finding new food sources. Here, we identify a spontaneous genetic change that increases exploration behavior and fitness of animals in laboratory environments. Interestingly, this genetic change is not a simple genetic change that deletes or changes the sequence of a protein product, but rather a complex structural variant that simultaneously duplicates the rcan-1 gene and also modifies its expression in a number of tissues. Our work demonstrates how a complex structural change can duplicate a gene, modify the DNA control regions that determine its cellular sites of action, and confer a fitness advantage that could lead to its spread in a population. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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34. Effects of the killer immunoglobulin–like receptor (KIR) polymorphisms on HIV acquisition: A meta-analysis.
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Chaisri, Suwit, Pabalan, Noel, Tabunhan, Sompong, Tharabenjasin, Phuntila, Sankuntaw, Nipaporn, and Leelayuwat, Chanvit
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META-analysis ,BONFERRONI correction ,ODDS ratio ,CONFIDENCE intervals ,CAUCASIAN race ,HIV - Abstract
Background: Genetic involvement of Killer Immunoglobulin-like Receptor (KIR) polymorphisms and Human Immunodeficiency Virus (HIV)-exposed seronegative (HESN) compared to HIV-infected (HIVI) individuals has been reported. However, inconsistency of the outcomes reduces precision of the estimates. A meta-analysis was applied to obtain more precise estimates of association. Methods: A multi-database literature search yielded thirteen case-control studies. Risks were expressed as odds ratios (ORs) and 95% confidence intervals (CIs) with significance set at a two-tailed P-value of ≤ 0.05. We used two levels of analyses: (1) gene content that included 13 KIR polymorphisms (2DL1-3, 2DL5A, 2DL5B, 2DS1-3, 2DS4F, 2DS4D, 2DS5, 3DL1 and 3DS1); and (2) 3DL1/S1 genotypes. Subgroup analysis was ethnicity-based (Caucasians, Asians and Africans). Outlier treatment was applied to heterogeneous effects which dichotomized the outcomes into pre-outlier (PRO) and post-outlier (PSO). Multiple comparisons were addressed with the Bonferroni correction. Results: We generated 52 and 18 comparisons from gene content and genotype analyses, respectively. Of the 70 comparisons, 13 yielded significant outcomes, two (indicating reduced risk) of which survived the Bonferroni correction (P
c ). These protective effects pointed to the Caucasian subgroup in 2DL3 (OR 0.19, 95% CI 0.09, 0.40, Pc < 10−3 ) and 3DS1S1 (OR 0.37, 95% CI 0.24, 0.56, Pc < 10−3 ). These two PSO outcomes yielded effects of increased magnitude and precision, as well as raised significance and deemed robust by sensitivity analysis. Of the two, the 2DL3 effect was improved with a test of interaction (Pc interaction < 10−4 ). Conclusion: Multiple meta-analytical treatments presented strong evidence of the protective effect (up to 81%) of the KIR polymorphisms (2DL3 and 3DS1S1) among Caucasians. The Asian and African outcomes were inconclusive due to the low number of studies. [ABSTRACT FROM AUTHOR]- Published
- 2019
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35. Origins of DNA replication.
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Ekundayo, Babatunde and Bleichert, Franziska
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DNA replication ,DNA synthesis ,CELL division ,DNA structure ,MOLECULAR biology ,NUCLEIC acids - Abstract
In all kingdoms of life, is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by prior to cell division to ensure each daughter cell receives the full complement of . DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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36. Identification of divergent Leishmania (Viannia) braziliensis ecotypes derived from a geographically restricted area through whole genome analysis.
- Author
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S. L. Figueiredo de Sá, Bruna, Rezende, Antonio M., Melo Neto, Osvaldo P. de, Brito, Maria Edileuza F. de, and Brandão Filho, Sinval P.
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HSP70 heat-shock proteins ,LEISHMANIA mexicana ,LEISHMANIA ,SINGLE nucleotide polymorphisms ,CUTANEOUS leishmaniasis ,COMPUTATIONAL biology - Abstract
Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis (CL) in Latin America, is characterized by major differences in basic biology in comparison with better-known Leishmania species. It is also associated with a high phenotypic and possibly genetic diversity that need to be more adequately defined. Here we used whole genome sequences to evaluate the genetic diversity of ten L. braziliensis isolates from a CL endemic area from Northeastern Brazil, previously classified by Multi Locus Enzyme Electrophoresis (MLEE) into ten distinct zymodemes. These sequences were first mapped using the L. braziliensis M2904 reference genome followed by identification of Single Nucleotide Polymorphisms (SNPs). A substantial level of diversity was observed when compared with the reference genome, with SNP counts ranging from ~95,000 to ~131,000 for the different isolates. When the genome data was used to infer relationship between isolates, those belonging to zymodemes Z72/Z75, recovered from forested environments, were found to cluster separately from the others, generally associated with more urban environments. Among the remaining isolates, those from zymodemes Z74/Z106 were also found to form a separate group. Phylogenetic analyses were also performed using Multi-Locus Sequence Analysis from genes coding for four metabolic enzymes used for MLEE as well as the gene sequence coding for the Hsp70 heat shock protein. All 10 isolates were firmly identified as L. braziliensis, including the zymodeme Z26 isolate previously classified as Leishmania shawi, with the clustering into three groups confirmed. Aneuploidy was also investigated but found in general restricted to chromosome 31, with a single isolate, from zymodeme Z27, characterized by extra copies for other chromosomes. Noteworthy, both Z72 and Z75 isolates are characterized by a much reduced heterozygosity. Our data is consistent with the existence of distinct evolutionary groups in the restricted area sampled and a substantial genetic diversity within L. braziliensis. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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37. Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches.
- Author
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Sanglard, Dominique
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DRUG resistance ,ANTIFUNGAL agents ,PHYTOPATHOGENIC fungi ,TARGETED drug delivery ,DRUG toxicity - Abstract
The article discusses antifungal resistance in plant and human fungal pathogens with a special focus on genome-wide studies. It mentions that resistance mechanisms can be grouped in three general principles including decrease of effective intracellular drug concentration, alterations of the drug target, and compensatory mechanisms which decrease drug toxicity.
- Published
- 2019
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38. Copy number variation in the susceptibility to systemic lupus erythematosus.
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Barbosa, Fernanda Bueno, Simioni, Milena, Wiezel, Cláudia Emília Vieira, Torres, Fábio Rossi, Molck, Miriam Coelho, Bonilla, Melvin M., de Araujo, Tânia Kawasaki, Donadi, Eduardo Antônio, Gil-da-Silva-Lopes, Vera Lúcia, Lemos, Bernardo, and Simões, Aguinaldo Luiz
- Subjects
SYSTEMIC lupus erythematosus ,DNA copy number variations ,AUTOANTIBODIES ,DELETION mutation ,POLYMERASE chain reaction - Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic component and etiology characterized by chronic inflammation and autoantibody production. The purpose of this study was to ascertain copy number variation (CNV) in SLE using a case-control design in an admixed Brazilian population. The whole-genome detection of CNV was performed using Cytoscan HD array in SLE patients and healthy controls. The best CNV candidates were then evaluated by quantitative real-time PCR in a larger cohort or validated using droplet digital PCR. Logistic regression models adjusted for sex and ancestry covariates was applied to evaluate the association between CNV with SLE susceptibility. The data showed a synergistic effect between the FCGR3B and ADAM3A loci with the presence of deletions in both loci significantly increasing the risk to SLE (5.9-fold) compared to the deletion in the single FCGR3B locus (3.6-fold). In addition, duplications in these genes were indeed more frequent in healthy subjects, suggesting that high FCGR3B/ADAM3A gene copy numbers are protective factors against to disease development. Overall, 21 rare CNVs were identified in SLE patients using a four-step pipeline created for identification of rare variants. Furthermore, heterozygous deletions overlapping the CFHR4, CFHR5 and HLA-DPB2 genes were described for the first time in SLE patients. Here we present the first genome-wide CNV study of SLE patients in a tri-hybrid population. The results show that novel susceptibility loci to SLE can be found once the distribution of structural variants is analyzed throughout the whole genome. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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39. Atypical leishmaniasis: A global perspective with emphasis on the Indian subcontinent.
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Thakur, Lovlesh, Singh, Kiran K., Shanker, Vinay, Negi, Ajeet, Jain, Aklank, Matlashewski, Greg, and Jain, Manju
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LEISHMANIASIS treatment ,PROTOZOAN diseases ,VISCERAL leishmaniasis ,LEISHMANIA mexicana ,PUBLIC health ,PREVENTION - Abstract
Background: Among the neglected tropical diseases, leishmaniasis continues to be prevalent in many tropical and subtropical countries despite international, national, and local efforts towards its control and elimination over the last decade. This warrants a critical evaluation of such factors as under-reporting, asymptomatic infections, post kala azar dermal leishmaniasis (PKDL) cases, and drug resistance. In this review, we highlight lesser-understood atypical presentations of the disease involving atypical parasite strains against a background of classical leishmaniasis with a focus on the Indian subcontinent. Methods and findings: A literature review based on endemic areas, the nature of disease manifestation, and underlying causative parasite was performed with data collected from WHO reports for each country. Searches on PubMed included the term ‘‘leishmaniasis” and “leishmaniasis epidemiology” alone and in combination with each of the endemic countries, Leishmania species, cutaneous, visceral, endemic, non-endemic, typical, classical, atypical, and unusual with no date limit and published in English up to September 2017. Our findings portray a scenario with a wider distribution of the disease in new endemic foci, with new discoveries of parasite-driven atypical disease manifestations in different regions of the world. Unlike the classical picture, some Leishmania species are associated with more than one disease presentation, e.g., the L. donovani complex, generally associated with the visceral form, is now also associated with a cutaneous disease presentation, while L. tropica species complex, known to cause cutaneous disease, can cause viscerotropic disease. This phenomenon points towards the discovery of novel parasite variants as etiologic agents of atypical disease manifestations and represents an excellent opportunity to identify and study genes that control disease virulence and tropism. Conclusions: The increased recognition of atypical leishmaniasis as an outcome of parasite variants has major implications for leishmaniasis control and elimination. Identifying molecular correlates of parasite isolates from distinct regions associated with different disease phenotypes is required to understand the current epidemiology of leishmaniasis in regions with atypical disease. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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40. Molecular assays for antimalarial drug resistance surveillance: A target product profile.
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Nsanzabana, Christian, Ariey, Frederic, Beck, Hans-Peter, Ding, Xavier C., Kamau, Edwin, Krishna, Sanjeev, Legrand, Eric, Lucchi, Naomi, Miotto, Olivo, Nag, Sidsel, Noedl, Harald, Roper, Cally, Rosenthal, Philip J., Schallig, Henk D. F. H., Taylor, Steve M., Volkman, Sarah K., and Gonzalez, Iveth J.
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ANTIMALARIALS ,ANTIPROTOZOAL agents ,MOLECULAR structure of antimalarials ,DRUG resistance ,MICROBIOLOGICAL assay - Abstract
Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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41. Genome-wide identification and analysis of the ALTERNATIVE OXIDASE gene family in diploid and hexaploid wheat.
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Brew-Appiah, Rhoda A. T., York, Zara B., Krishnan, Vandhana, Roalson, Eric H., and Sanguinet, Karen A.
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GENOME statistics ,OXIDASES ,DIPLOIDY ,WHEAT ,COMPUTATIONAL biology - Abstract
A comprehensive understanding of wheat responses to environmental stress will contribute to the long-term goal of feeding the planet. ALERNATIVE OXIDASE (AOX) genes encode proteins involved in a bypass of the electron transport chain and are also known to be involved in stress tolerance in multiple species. Here, we report the identification and characterization of the AOX gene family in diploid and hexaploid wheat. Four genes each were found in the diploid ancestors Triticum urartu, and Aegilops tauschii, and three in Aegilops speltoides. In hexaploid wheat (Triticum aestivum), 20 genes were identified, some with multiple splice variants, corresponding to a total of 24 proteins for those with observed transcription and translation. These proteins were classified as AOX1a, AOX1c, AOX1e or AOX1d via phylogenetic analysis. Proteins lacking most or all signature AOX motifs were assigned to putative regulatory roles. Analysis of protein-targeting sequences suggests mixed localization to the mitochondria and other organelles. In comparison to the most studied AOX from Trypanosoma brucei, there were amino acid substitutions at critical functional domains indicating possible role divergence in wheat or grasses in general. In hexaploid wheat, AOX genes were expressed at specific developmental stages as well as in response to both biotic and abiotic stresses such as fungal pathogens, heat and drought. These AOX expression patterns suggest a highly regulated and diverse transcription and expression system. The insights gained provide a framework for the continued and expanded study of AOX genes in wheat for stress tolerance through breeding new varieties, as well as resistance to AOX-targeted herbicides, all of which can ultimately be used synergistically to improve crop yield. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
42. A population genomic characterization of copy number variation in the opportunistic fungal pathogen Aspergillus fumigatus.
- Author
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Zhao, Shu and Gibbons, John G.
- Subjects
METAGENOMICS ,ASPERGILLUS fumigatus ,PATHOGENIC microorganisms ,MYCOSES ,ASPERGILLUS ,GENETICS ,PHYSIOLOGY - Abstract
Aspergillus fumigatus is a potentially deadly opportunistic fungal pathogen. Molecular studies have shaped our understanding of the genes, proteins, and molecules that contribute to A. fumigatus pathogenicity, but few studies have characterized genome-wide patterns of genetic variation at the population level. Of A. fumigatus genomic studies to-date, most focus mainly on single nucleotide polymorphisms and large structural variants, while overlooking the contribution of copy number variation (CNV). CNV is a class of small structural variation defined as loci that vary in their number of copies between individuals due to duplication, gain, or deletion. CNV can influence phenotype, including fungal virulence. In the present study, we characterized the population genomic patterns of CNV in a diverse collection of 71 A. fumigatus isolates using publicly available sequencing data. We used genome-wide single nucleotide polymorphisms to infer the population structure of these isolates and identified three populations consisting of at least 8 isolates. We then computationally predicted genome-wide CNV profiles for each isolate and conducted analyses at the species-, population-, and individual levels. Our results suggest that CNV contributes to genetic variation in A. fumigatus, with ~10% of the genome being CN variable. Our analysis indicates that CNV is non-randomly distributed across the A. fumigatus genome, and is overrepresented in subtelomeric regions. Analysis of gene ontology categories in genes that overlapped CN variants revealed an enrichment of genes related to transposable element and secondary metabolism functions. We further identified 72 loci containing 33 genes that showed divergent copy number profiles between the three A. fumigatus populations. Many of these genes encode proteins that interact with the cell surface or are involved in pathogenicity. Our results suggest that CNV is an important source of genetic variation that could account for some of the phenotypic differences between A. fumigatus populations and isolates. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
43. Sequence characteristics define trade-offs between on-target and genome-wide off-target hybridization of oligoprobes.
- Author
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Matveeva, Olga V., Ogurtsov, Aleksey Y., Nazipova, Nafisa N., and Shabalina, Svetlana A.
- Subjects
GENOMES ,NUCLEOTIDE sequence ,NUCLEOTIDES ,NUCLEOTIDE sequencing ,BINDING energy - Abstract
Off-target oligoprobe’s interaction with partially complementary nucleotide sequences represents a problem for many bio-techniques. The goal of the study was to identify oligoprobe sequence characteristics that control the ratio between on-target and off-target hybridization. To understand the complex interplay between specific and genome-wide off-target (cross-hybridization) signals, we analyzed a database derived from genomic comparison hybridization experiments performed with an Affymetrix tiling array. The database included two types of probes with signals derived from (i) a combination of specific signal and cross-hybridization and (ii) genomic cross-hybridization only. All probes from the database were grouped into bins according to their sequence characteristics, where both hybridization signals were averaged separately. For selection of specific probes, we analyzed the following sequence characteristics: vulnerability to self-folding, nucleotide composition bias, numbers of G nucleotides and GGG-blocks, and occurrence of probe’s k-mers in the human genome. Increases in bin ranges for these characteristics are simultaneously accompanied by a decrease in hybridization specificity—the ratio between specific and cross-hybridization signals. However, both averaged hybridization signals exhibit growing trends along with an increase of probes’ binding energy, where the hybridization specific signal increases significantly faster in comparison to the cross-hybridization. The same trend is evident for the S function, which serves as a combined evaluation of probe binding energy and occurrence of probe’s k-mers in the genome. Application of S allows extracting a larger number of specific probes, as compared to using only binding energy. Thus, we showed that high values of specific and cross-hybridization signals are not mutually exclusive for probes with high values of binding energy and S. In this study, the application of a new set of sequence characteristics allows detection of probes that are highly specific to their targets for array design and other bio-techniques that require selection of specific probes. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
44. AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica.
- Author
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Nakada-Tsukui, Kumiko, Sekizuka, Tsuyoshi, Sato-Ebine, Emi, Escueta-de Cadiz, Aleyla, Ji, Dar-der, Tomii, Kentaro, Kuroda, Makoto, and Nozaki, Tomoyoshi
- Subjects
AMEBIASIS ,ENTAMOEBA histolytica ,MICROBIAL virulence ,HOSTS (Biology) ,COMPARATIVE genomics - Abstract
The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-nduced ene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
45. CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1.
- Author
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Ishemgulova, Aygul, Hlaváčová, Jana, Majerová, Karolina, Butenko, Anzhelika, Lukeš, Julius, Votýpka, Jan, Volf, Petr, and Yurchenko, Vyacheslav
- Subjects
CRISPRS ,LEISHMANIA mexicana ,VISCERAL leishmaniasis ,GENETICS - Abstract
Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM.22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM.22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM.22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM.22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
46. eGARD: Extracting associations between genomic anomalies and drug responses from text.
- Author
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Mahmood, A. S. M. Ashique, Rao, Shruti, McGarvey, Peter, Wu, Cathy, Madhavan, Subha, and Vijay-Shanker, K.
- Subjects
DRUG therapy ,CANCER treatment ,INDIVIDUALIZED medicine ,TEXT mining ,INFORMATION technology - Abstract
Tumor molecular profiling plays an integral role in identifying genomic anomalies which may help in personalizing cancer treatments, improving patient outcomes and minimizing risks associated with different therapies. However, critical information regarding the evidence of clinical utility of such anomalies is largely buried in biomedical literature. It is becoming prohibitive for biocurators, clinical researchers and oncologists to keep up with the rapidly growing volume and breadth of information, especially those that describe therapeutic implications of biomarkers and therefore relevant for treatment selection. In an effort to improve and speed up the process of manually reviewing and extracting relevant information from literature, we have developed a natural language processing (NLP)-based text mining (TM) system called eGARD (extracting Genomic Anomalies association with Response to Drugs). This system relies on the syntactic nature of sentences coupled with various textual features to extract relations between genomic anomalies and drug response from MEDLINE abstracts. Our system achieved high precision, recall and F-measure of up to 0.95, 0.86 and 0.90, respectively, on annotated evaluation datasets created in-house and obtained externally from PharmGKB. Additionally, the system extracted information that helps determine the confidence level of extraction to support prioritization of curation. Such a system will enable clinical researchers to explore the use of published markers to stratify patients upfront for ‘best-fit’ therapies and readily generate hypotheses for new clinical trials. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
47. Integrative analysis of genome-wide gene copy number changes and gene expression in non-small cell lung cancer.
- Author
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Jabs, Verena, Edlund, Karolina, König, Helena, Grinberg, Marianna, Madjar, Katrin, Rahnenführer, Jörg, Ekman, Simon, Bergkvist, Michael, Holmberg, Lars, Ickstadt, Katja, Botling, Johan, Hengstler, Jan G., and Micke, Patrick
- Subjects
NON-small-cell lung carcinoma ,DNA copy number variations ,GENE expression ,MESSENGER RNA ,GENOMES ,GENETICS - Abstract
Non-small cell lung cancer (NSCLC) represents a genomically unstable cancer type with extensive copy number aberrations. The relationship of gene copy number alterations and subsequent mRNA levels has only fragmentarily been described. The aim of this study was to conduct a genome-wide analysis of gene copy number gains and corresponding gene expression levels in a clinically well annotated NSCLC patient cohort (n = 190) and their association with survival. While more than half of all analyzed gene copy number-gene expression pairs showed statistically significant correlations (10,296 of 18,756 genes), high correlations, with a correlation coefficient >0.7, were obtained only in a subset of 301 genes (1.6%), including KRAS, EGFR and MDM2. Higher correlation coefficients were associated with higher copy number and expression levels. Strong correlations were frequently based on few tumors with high copy number gains and correspondingly increased mRNA expression. Among the highly correlating genes, GO groups associated with posttranslational protein modifications were particularly frequent, including ubiquitination and neddylation. In a meta-analysis including 1,779 patients we found that survival associated genes were overrepresented among highly correlating genes (61 of the 301 highly correlating genes, FDR adjusted p<0.05). Among them are the chaperone CCT2, the core complex protein NUP107 and the ubiquitination and neddylation associated protein CAND1. In conclusion, in a comprehensive analysis we described a distinct set of highly correlating genes. These genes were found to be overrepresented among survival-associated genes based on gene expression in a large collection of publicly available datasets. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
48. Whole-genome sequencing reveals mutational landscape underlying phenotypic differences between two widespread Chinese cattle breeds.
- Author
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Xu, Yao, Jiang, Yu, Shi, Tao, Cai, Hanfang, Lan, Xianyong, Zhao, Xin, Plath, Martin, and Chen, Hong
- Subjects
CATTLE breeding ,CATTLE genetics ,CATTLE genome mapping ,NUCLEOTIDE sequencing ,PHENOTYPES - Abstract
Whole-genome sequencing provides a powerful tool to obtain more genetic variability that could produce a range of benefits for cattle breeding industry. Nanyang (Bos indicus) and Qinchuan (Bos taurus) are two important Chinese indigenous cattle breeds with distinct phenotypes. To identify the genetic characteristics responsible for variation in phenotypes between the two breeds, in the present study, we for the first time sequenced the genomes of four Nanyang and four Qinchuan cattle with 10 to 12 fold on average of 97.86% and 98.98% coverage of genomes, respectively. Comparison with the Bos_taurus_UMD_3.1 reference assembly yielded 9,010,096 SNPs for Nanyang, and 6,965,062 for Qinchuan cattle, 51% and 29% of which were novel SNPs, respectively. A total of 154,934 and 115,032 small indels (1 to 3 bp) were found in the Nanyang and Qinchuan genomes, respectively. The SNP and indel distribution revealed that Nanyang showed a genetically high diversity as compared to Qinchuan cattle. Furthermore, a total of 2,907 putative cases of copy number variation (CNV) were identified by aligning Nanyang to Qinchuan genome, 783 of which (27%) encompassed the coding regions of 495 functional genes. The gene ontology (GO) analysis revealed that many CNV genes were enriched in the immune system and environment adaptability. Among several CNV genes related to lipid transport and fat metabolism, Lepin receptor gene (LEPR) overlapping with CNV_1815 showed remarkably higher copy number in Qinchuan than Nanyang (log
2 (ratio) = -2.34988; P value = 1.53E-102). Further qPCR and association analysis investigated that the copy number of the LEPR gene presented positive correlations with transcriptional expression and phenotypic traits, suggesting the LEPR CNV may contribute to the higher fat deposition in muscles of Qinchuan cattle. Our findings provide evidence that the distinct phenotypes of Nanyang and Qinchuan breeds may be due to the different genetic variations including SNPs, indels and CNV. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
49. Loss of SLC9A3 decrease CFTR protein and causes obstructed azoospermia in mice.
- Author
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Wang, Ya-Yun, Lin, Ying-Hung, Wu, Yi-No, Chen, Yen-Lin, Lin, Yung-Chih, Cheng, Chiao-Yin, and Chiang, Han-Sun
- Subjects
CYSTIC fibrosis transmembrane conductance regulator ,GENETIC mutation ,KNOCKOUT mice ,EPIDIDYMIS ,GENE expression - Abstract
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major CFTR mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (SLC9A3) gene. SLC9A3 is a Na
+ /H+ exchanger, and depleted Slc9a3 in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of Slc9a3 knockout mice. Adult Slc9a3-/- mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, Slc9a3-/- mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in Slc9a3–/– epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The Slc9a3-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry SLC9A3 deletion but without major CFTR mutations. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
50. The temporal dynamics of chromosome instability in ovarian cancer cell lines and primary patient samples.
- Author
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Penner-Goeke, Signe, Lichtensztejn, Zelda, Neufeld, Megan, Ali, Jennifer L., Altman, Alon D., Nachtigal, Mark W., and McManus, Kirk J.
- Subjects
OVARIAN epithelial cancer ,CELL lines ,CHROMOSOMES ,DRUG resistance ,ASCITES tumors ,CANCER relapse - Abstract
Epithelial ovarian cancer (EOC) is the most prevalent form of ovarian cancer and has the highest mortality rate. Novel insight into EOC is required to minimize the morbidity and mortality rates caused by recurrent, drug resistant disease. Although numerous studies have evaluated genome instability in EOC, none have addressed the putative role chromosome instability (CIN) has in disease progression and drug resistance. CIN is defined as an increase in the rate at which whole chromosomes or large parts thereof are gained or lost, and can only be evaluated using approaches capable of characterizing genetic or chromosomal heterogeneity within populations of cells. Although CIN is associated with numerous cancer types, its prevalence and dynamics in EOC is unknown. In this study, we assessed CIN within serial samples collected from the ascites of five EOC patients, and in two well-established ovarian cancer cell models of drug resistance (PEO1/4 and A2780s/cp). We quantified and compared CIN (as measured by nuclear areas and CIN Score (CS) values) within and between serial samples to glean insight into the association and dynamics of CIN within EOC, with a particular focus on resistant and recurrent disease. Using quantitative, single cell analyses we determined that CIN is associated with every sample evaluated and further show that many EOC samples exhibit a large degree of nuclear size and CS value heterogeneity. We also show that CIN is dynamic and generally increases within resistant disease. Finally, we show that both drug resistance models (PEO1/4 and A2780s/cp) exhibit heterogeneity, albeit to a much lesser extent. Surprisingly, the two cell line models exhibit remarkably similar levels of CIN, as the nuclear areas and CS values are largely overlapping between the corresponding paired lines. Accordingly, these data suggest CIN may represent a novel biomarker capable of monitoring changes in EOC progression associated with drug resistance. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
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