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Background and Aim: Among the cardiovascular diseases (CVDs), heart failure, hypertension, and myocardial infarction are associated with the greatest number of disability‐adjusted life years due to lifestyle changes and the failure of therapeutic approaches, especially the one‐size‐fits‐all interventions. As a result, there has been advances in defining genetic variants responsible for different responses to cardiovascular drugs such as antiplatelets, anticoagulants, statins, and beta‐blockers, which has led to their usage in guiding treatment plans. This study comprehensively reviews the current state‐of‐the‐art potential of pharmacogenomics in dramatically altering CVD treatment. It stresses the applicability of pharmacogenomic technology, the threats associated with its adoption in the clinical setting, and proffers relevant solutions. Methods: Literature search strategies were used to retrieve articles from various databases: PubMed, Google Scholar, and EBSCOhost. Articles with information relevant to pharmacogenomics, DNA variants, cardiovascular diseases, sequencing techniques, and drug responses were reviewed and analyzed. Results: DNA‐based technologies such as next generation sequencing, whole genome sequencing, whole exome sequencing, and targeted segment sequencing can identify variants in the human genome. This has played a substantial role in identifying different genetic variants governing the poor response and adverse effects associated with cardiovascular drugs. Thus, this has reduced patients' number of emergency visits and hospitalization. Conclusion: Despite the emergence of pharmacogenomics, its implementation has been threatened by factors including patient compliance and a low adoption rate by clinicians. Education and training programs targeting both healthcare professionals and patients should be established to increase the acceptance and application of the emerging pharmacogenomic technologies in reducing the burden of CVDs. [ABSTRACT FROM AUTHOR]

Abstract Background and Aim Among the cardiovascular diseases (CVDs), heart failure, hypertension, and myocardial infarction are associated with the greatest number of disability‐adjusted life years due to lifestyle changes and the failure of therapeutic approaches, especially the one‐size‐fits‐all interventions. As a result, there has been advances in defining genetic variants responsible for different responses to cardiovascular drugs such as antiplatelets, anticoagulants, statins, and beta‐blockers, which has led to their usage in guiding treatment plans. This study comprehensively reviews the current state‐of‐the‐art potential of pharmacogenomics in dramatically altering CVD treatment. It stresses the applicability of pharmacogenomic technology, the threats associated with its adoption in the clinical setting, and proffers relevant solutions. Methods Literature search strategies were used to retrieve articles from various databases: PubMed, Google Scholar, and EBSCOhost. Articles with information relevant to pharmacogenomics, DNA variants, cardiovascular diseases, sequencing techniques, and drug responses were reviewed and analyzed. Results DNA‐based technologies such as next generation sequencing, whole genome sequencing, whole exome sequencing, and targeted segment sequencing can identify variants in the human genome. This has played a substantial role in identifying different genetic variants governing the poor response and adverse effects associated with cardiovascular drugs. Thus, this has reduced patients' number of emergency visits and hospitalization. Conclusion Despite the emergence of pharmacogenomics, its implementation has been threatened by factors including patient compliance and a low adoption rate by clinicians. Education and training programs targeting both healthcare professionals and patients should be established to increase the acceptance and application of the emerging pharmacogenomic technologies in reducing the burden of CVDs.

Simple Summary: Using the Nanopore platform, we determined the whole genome sequence of an H3N8 equine influenza virus, identified from a 2005 outbreak in Apulia, Italy. The virus was closely related (>99% at the nucleotide level) in all the genome segments to viruses identified in Poland in 2005–2008 and was an inter-lineage multi-reassortant between Florida 2 and Eurasian viruses. Massive sequencing techniques have allowed us to develop straightforward approaches for the whole genome sequencing of viruses, including influenza viruses, generating information that is useful for improving the levels and dimensions of data analysis, even for archival samples. Using the Nanopore platform, we determined the whole genome sequence of an H3N8 equine influenza virus, identified from a 2005 outbreak in Apulia, Italy, whose origin had remained epidemiologically unexplained. The virus was tightly related (>99% at the nucleotide level) in all the genome segments to viruses identified in Poland in 2005–2008 and it was seemingly introduced locally with horse trading for the meat industry. In the phylogenetic analysis based on the eight genome segments, strain ITA/2005/horse/Bari was found to cluster with sub-lineage Florida 2 in the HA and M genes, whilst in the other genes it clustered with strains of the Eurasian lineage, revealing a multi-reassortant nature. [ABSTRACT FROM AUTHOR]

Multiple types of non-canonical nucleic acid structures play essential roles in DNA recombination and replication, transcription, and genomic instability and have been associated with several human diseases. Thus, an increasing number of experimental and bioinformatics methods have been developed to identify these structures. To date, most reviews have focused on the features of non-canonical DNA/RNA structure formation, experimental approaches to mapping these structures, and the association of these structures with diseases. In addition, two reviews of computational algorithms for the prediction of non-canonical nucleic acid structures have been published. One of these reviews focused only on computational approaches for G4 detection until 2020. The other mainly summarized the computational tools for predicting cruciform, H-DNA and Z-DNA, in which the algorithms discussed were published before 2012. Since then, several experimental and computational methods have been developed. However, a systematic review including the conformation, sequencing mapping methods and computational prediction strategies for these structures has not yet been published. The purpose of this review is to provide an updated overview of conformation, current sequencing technologies and computational identification methods for non-canonical nucleic acid structures, as well as their strengths and weaknesses. We expect that this review will aid in understanding how these structures are characterised and how they contribute to related biological processes and diseases. [ABSTRACT FROM AUTHOR]

Massive sequencing techniques have allowed us to develop straightforward approaches for the whole genome sequencing of viruses, including influenza viruses, generating information that is useful for improving the levels and dimensions of data analysis, even for archival samples. Using the Nanopore platform, we determined the whole genome sequence of an H3N8 equine influenza virus, identified from a 2005 outbreak in Apulia, Italy, whose origin had remained epidemiologically unexplained. The virus was tightly related (>99% at the nucleotide level) in all the genome segments to viruses identified in Poland in 2005–2008 and it was seemingly introduced locally with horse trading for the meat industry. In the phylogenetic analysis based on the eight genome segments, strain ITA/2005/horse/Bari was found to cluster with sub-lineage Florida 2 in the HA and M genes, whilst in the other genes it clustered with strains of the Eurasian lineage, revealing a multi-reassortant nature.

Genome-wide association studies (GWAS) have commonly been used to identify candidate genes that control economically important traits in livestock. Our objective was to detect potential candidate genes associated mainly with muscle development traits related to dimension of hindquarter in cattle. A next generation sequencing (NGS) dataset to imputed to 12 million single nucleotide polymorphisms (SNPs) (from 1252 Simmental beef cattle) were used to search for genes affecting hindquarter traits using a linear, mixed model approach. We also used haplotype and linkage disequilibrium blocks to further support our identifications. We identified 202 significant SNPs in the bovine BTA4 chromosome region associated with width of hind leg, based on a stringent statistical threshold (p = 0.05/ effective number of SNPs identified). After exploring the region around these SNPs, we found candidate genes that were potentially related to the associated markers. More importantly, we identified a region of approximately 280 Kb on the BTA4 chromosome that harbored several muscle specific candidate genes, genes to be in a potential region for muscle development. However, we also found candidate gene SLC13A1 on BTA4, which seems to be associated with bone disorders (such as chondrodysplasia) in Simmental beef cattle. [ABSTRACT FROM AUTHOR]

The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone–derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population. [ABSTRACT FROM AUTHOR]

Limited translational genomic research data have been reported on the application of exome sequencing and parallel gene testing for preconception carrier screening (PCS). Here, we present individual-level data from a large PCS program in which exome sequencing was routinely performed on either gamete donors (5,845) or infertile patients (8,280) undergoing in vitro fertilization (IVF) treatment without any known family history of inheritable genetic conditions. Individual-level data on pathogenic variants were used to define conditions for PCS based on criteria for severity, penetrance, inheritance pattern, and age of onset. Fetal risk was defined based on actual carrier frequency data accounting for the specific inheritance pattern (fetal disease risk, FDR). In addition, large-scale application of exome sequencing for PCS allowed a deep investigation of the incidence of medically actionable secondary findings in this population. Exome sequencing achieved remarkable clinical sensitivity for reproductive risk of highly penetrant childhood-onset disorders (1/337 conceptions) through analysis of 114 selected gene-condition pairs. A significant contribution to fetal disease risk was observed for rare (carrier rate < 1:100) and X-linked conditions (16.7% and 41.2% of total FDR, respectively). Subgroup analysis of 776 IVF couples identified 37 at increased reproductive risk (4.8%; 95% CI = 3.4–6.5). Further, two additional couples had increased risk for very rare conditions when both members of a parental pair were treated as a unit and the search was extended to the entire exome. About 2.3% of participants showed at least one pathogenic variant for genes included in the updated American College of Medical Genetics and Genomics v2.0 list of secondary findings. Gamete donors and IVF couples showed similar carrier burden for both carrier screening and secondary findings, indicating no causal relationship to fertility. These translational research data will facilitate development of more effective PCS strategies that maximize clinical sensitivity with minimal counterproductive effects. [ABSTRACT FROM AUTHOR]

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome. [ABSTRACT FROM AUTHOR]

Two novel and related pathogenic variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene were structurally and functionally characterized. These alterations have not been previously described in literature. Two patients with diagnosis of Cystic Fibrosis (CF) based on the presence of one mutated allele, p.Phe508del, pathological sweat test and clinical symptoms were studied. To complete the genotypes of both patients, an extensive genetic and functional analysis of the CFTR gene was performed. Extensive genetic characterization confirmed the presence of p.Phe508del pathogenic variant and revealed, in both patients, the presence of an insertion of part of intron 10 in intron 9 of the CFTR gene, within the (TG)m repeat, with a variable poly-T stretch. The molecular lesions resulted to be very similar in both patients, with only a difference in the number of T in the poly-T stretch. The functional characterization at RNA level revealed a complete anomalous splicing, without exon 10, from the allele with the insertion of both patients. Consequently, the alleles with the insertions are expected not to contribute to the formation of a functional CFTR protein. Molecular and functional features of these alterations are compatible with the definition of novel CF-causing variants of the CFTR gene. This also allowed the completion of the genetic characterization of both patients. [ABSTRACT FROM AUTHOR]

Expression quantitative trait loci (eQTL) provide insight on transcription regulation and illuminate the molecular basis of phenotypic outcomes. High-throughput RNA sequencing (RNA-seq) is becoming a popular technique to measure gene expression abundance. Traditional eQTL mapping methods for microarray expression data often assume the expression data follow a normal distribution. As a result, for RNA-seq data, total read count measurements can be normalized by normal quantile transformation in order to fit the data using a linear regression. Other approaches model the total read counts using a negative binomial regression. While these methods work well for common variants (minor allele frequencies > 5% or 1%), an extension of existing methodology is needed to accommodate a collection of rare variants in RNA-seq data. Here, we examine 2 approaches that are direct applications of existing methodology and apply these approaches to RNAseq studies: 1) collapsing the rare variants in the region and using either negative binomial regression or Poisson regression and 2) using the normalized read counts with the Sequence Kernel Association Test (SKAT), the burden test for SKAT (SKAT-Burden), or an optimal combination of these two tests (SKAT-O). We evaluated these approaches via simulation studies under numerous scenarios and applied these approaches to the 1,000 Genomes Project. [ABSTRACT FROM AUTHOR]

Lutzomyia longipalpis is the main vector of Leishmania infantum, the causative agent of visceral leishmaniasis in the Neotropical realm. Its taxonomic status has been widely discussed once it encompasses a complex of species. The knowledge about the genetic structure of insect vector populations helps the elucidation of components and interactions of the disease ecoepidemiology. Thus, the objective of this study was to genotypically analyze populations of the Lu. longipalpis complex from a macrogeographic perspective using Next Generation Sequencing. Polymorphism analysis of three molecular markers was used to access the levels of population genetic structure among nine different populations of sand flies. Illumina Amplicon Sequencing Protocol® was used to identify possible polymorphic sites. The library was sequenced on paired-end Illumina MiSeq platform. Significant macrogeographical population differentiation was observed among Lu. longipalpis populations via PCA and DAPC analyses. Our results revealed that populations of Lu. longipalpis from the nine municipalities were grouped into three clusters. In addition, it was observed that the levels of Lu. longipalpis population structure could be associated with distance isolation. This new sequencing method allowed us to study different molecular markers after a single sequencing run, and to evaluate population and inter-species differences on a macrogeographic scale. [ABSTRACT FROM AUTHOR]

Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing. [ABSTRACT FROM AUTHOR]

Background: RNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5–35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data. Methods: We describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization. Results: We demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance. Conclusions: The approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis. [ABSTRACT FROM AUTHOR]

Background: Toxigenic Vibrio cholerae resides in aquatic reservoirs of cholera-endemic areas mostly in a dormant form known as conditionally viable environmental cells (CVEC) in which the bacteria remain embedded in an exopolysaccharide matrix, and fail to grow in routine bacteriological culture. The CVEC can be resuscitated by supplementing culture media with either of two autoinducers CAI-1 and AI-2, which are signal molecules controlling quorum sensing, a regulatory network of bacterial gene expression dependent on cell density. This study investigated possible existence of variant strains that overproduce AIs, sufficient to resuscitate CVEC in environmental waters. Methods: Environmental V. cholerae isolates and Tn insertion mutants of a V. cholerae strain C6706 were screened for production of AIs using bioluminescent reporter strains. Relevant mutations in environmental strains which overproduced AI-2 were characterized by nucleotide sequencing and genetic complementation studies. Effect of AIs produced in culture supernatants of relevant strains on reactivation of CVEC in water was determined by resuscitation assays. Results: Two of 54 environmental V. cholerae isolates were found to overproduce AI-2. Screening of a Tn-insertion library of V. cholerae strain C6706, identified a mutant which overproduced AI-2, and carried Tn insertion in the cqsS gene. Nucleotide sequencing also revealed mutations inactivating the cqsS gene in environmental isolates which overproduced AI-2, and this property was reversed when complemented with a wild type cqsS gene. Culture of river water samples supplemented with spent medium of these mutants resuscitated dormant V. cholerae cells in water. Significance: V. cholerae strains with inactivated cqsS gene may offer a convenient source of AI-2 in enhanced assays for monitoring bacteriological quality of water. The results also suggest a potential role of naturally occurring cqsS mutants in the environmental biology of V. cholerae. Furthermore, similar phenomenon may have relevance in the ecology of other waterborne bacterial pathogens beyond V. cholerae. [ABSTRACT FROM AUTHOR]

RNA contains various chemical modifications, among which N6-methyladenosine (m6A) is the most prevalent modified nucleotide in eukaryotic mRNA. Emerging evidence suggests that m6A plays an important role in regulating a variety of cellular functions by controlling mRNA processing, translation and degradation. Because m6A is not detectable by standard chemical modification-based approaches, immunological methods, such as ELISA, immunoblotting, immunohistochemistry, m6A RNA immunoprecipitation sequencing and m6A individual-nucleotide resolution cross-linking and immunoprecipitation, have been employed to detect m6A in RNA. Although the most important factor determining the success of these methods is the integrity of highly specific antibodies against m6A, the development of m6A-specific monoclonal antibodies has been challenging. We developed anti-m6A monoclonal antibodies using our recently developed single cell-based monoclonal antibody production system. The binding of one selected antibody, #B1-3, to RNA oligoribonucleotide containing a single m6A had an equilibrium dissociation constant of 6.5 nM, and this antibody exhibited negligible binding to oligoribonucleotides containing a single N1-methyladenosine and unmodified adenosine. The binding was competed by the addition of increasing concentrations of N6-methyl-ATP but not N1-methyl-ATP or ATP. Furthermore, this mAb specifically crosslinked m6A-containing oligoribonucleotide by ultraviolet light, resulting in the induction of cDNA truncation at m6A position. These results show the feasibility of using the validated m6A monoclonal antibody for the specific detection of m6A in RNA. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is a newly emerged swine enteropathogenic coronavirus affecting pigs of all ages and causing diarrhea problems. Research findings indicate that PDCoV has evolved strategies to escape innate immune response in host cells, but mechanism of PDCoV in innate immune modulation is not well understood. In this study, we report our findings on identifying the alterations of host cell innate immune response affected by PDCoV infection and exploring the gene expression profiles of PK-15 cells at 0, 24, and 36 h PDCoV post infection by RNA sequencing. A total of 3,762 and 560 differentially expressed genes (DEGs) were screened by comparison of uninfected PK-15 cells and infected PK-15 cells at 24 h post infection (hpi) (INF_24h versus NC), and also comparison of infected PK-15 cells between 24 and 36 hpi (INF_36h versus INF_24h), which included 156 and 23 porcine innate immune-related genes in the DEGs of INF_24h versus NC and INF_36h versus INF_24h, respectively. Gene Ontology function classification and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analysis were performed based on the DEGs that exhibited the same expression tendencies with most of the innate immune-associated genes among these PK-15 cell samples described above. The enrichment results indicated that extensive gene functions and signaling pathways including innate immune-associated functions and pathways were affected by PDCoV infection. Particularly, 4 of 5 innate immune signaling pathways, which were primarily affected by PDCoV, played important roles in I-IFN’s antiviral function in innate immune response. Additionally, 16 of the host cell endogenous miRNAs were predicted as potential contributors to the modulation of innate immune response affected by PDCoV. Our research findings indicated that the innate immune-associated genes and signaling pathways in PK-15 cells could be modified by the infection of PDCoV, which provides a fundamental foundation for further studies to better understand the mechanism of PDCoV infections, so as to effectively control and prevent PDCoV-induced swine diarrheal disease outbreaks. [ABSTRACT FROM AUTHOR]

Characterization of Human Endogenous Retrovirus (HERV) expression within the transcriptomic landscape using RNA-seq is complicated by uncertainty in fragment assignment because of sequence similarity. We present Telescope, a computational software tool that provides accurate estimation of transposable element expression (retrotranscriptome) resolved to specific genomic locations. Telescope directly addresses uncertainty in fragment assignment by reassigning ambiguously mapped fragments to the most probable source transcript as determined within a Bayesian statistical model. We demonstrate the utility of our approach through single locus analysis of HERV expression in 13 ENCODE cell types. When examined at this resolution, we find that the magnitude and breadth of the retrotranscriptome can be vastly different among cell types. Furthermore, our approach is robust to differences in sequencing technology, and demonstrates that the retrotranscriptome has potential to be used for cell type identification. We compared our tool with other approaches for quantifying TE expression, and found that Telescope has the greatest resolution, as it estimates expression at specific TE insertions rather than at the TE subfamily level. Telescope performs highly accurate quantification of the retrotranscriptomic landscape in RNA-seq experiments, revealing a differential complexity in the transposable element biology of complex systems not previously observed. Telescope is available at . [ABSTRACT FROM AUTHOR]

Angiostrongyliasis is a parasitic disease caused by nematodes of the genus Angiostrongylus. Distribution of this worm corresponds to the dispersal of its main intermediate host, the giant African land snail Achatina fulica. Genetic characterization can help identify parasitic pathogens and control the spreading of disease. The present study describes infection of A. fulica by Angiostrongylus, and provides a genetic outlook based on sequencing of specific regions. We collected 343 land snails from 22 provinces across six regions of Thailand between May 2017 and July 2018. Artificial digestion and Baermann’s technique were employed to isolate Angiostrongylus larvae. The worm and its intermediate host were identified by sequencing with specific nucleotide regions. Phylogenetic tree was constructed to evaluate the relationship with other isolates. A. fulica from Chaiyaphum province was infected with A. cantonensis, whereas snails collected from Phrae and Chiang Rai provinces were infected with A. malaysiensis. The maximum likelihood tree based on 74 A. fulica COI sequences revealed monophyletic groups and identified two haplotypes: AF1 and AF2. Only AF1, which is distributed in all regions of Thailand, harbored the larvae of A. cantonensis and A. malaysiensis. Two mitochondrial genes (COI and cytb) and two nuclear regions (ITS2 and SSU rRNA) were sequenced in 41 Angiostrongylus specimens. The COI gene indicated that A. cantonensis was closely related to the AC10 haplotype; whereas the cytb gene revealed two new haplotypes: AC19 and AC20. SSU rRNA was useful for the identification of A. cantonensis; whereas ITS2 was a good genetic marker for differentiating between A. cantonensis and A. malaysiensis. This study provides genetic information about the parasite Angiostrongylus and its snail intermediate host. The data in this work may be useful for further study on the identification of Angiostrongylus spp., the genetic relationship between intermediate host and parasite, and control of parasites. [ABSTRACT FROM AUTHOR]

Our research group has showed that the LIM homeobox transcription factor 1 alpha (LMX1A) is inactivated in gastric cancers. Overexpression of LMX1A inhibits tumor growth. However, the mechanisms remains unclear. Considering LMX1A as a transcription factor, a comparison of RNA-seq between gastric cancer cells (GCCs) and GCCs with LMX1A overexpressed was performed to identify genes transcriptionally activated by LMX1A. Among the potential LMX1A target genes, angiopoietin-like 4 (ANGPTL4) has been reported to be an important tumor suppressor and thus was selected for further validation and research. Both LMX1A and ANGPTL4 showed downregulated expression in gastric cancer samples. More importantly, the expression of LMX1A is positively correlated with ANGPTL4, without including other family members in gastric cancer cell lines. What’s more, knockdown of ANGPTL4 rescued the tumor suppressive phenotype of LMX1A overexpression, which indicated that LMX1A upregulates ANGPTL4 to exert its role. Mechanistically, we found that LMX1A inhibited the expression of the oncogene C-Myc, which is alleviated by ANGPTL4 knockdown. In general, our results showed that LMX1A exerts its tumor suppressive role by activating ANGPTL4 to inhibit C-Myc. [ABSTRACT FROM AUTHOR]

Telomeres are nucleo-protein structures that protect the ends of eukaryotic chromosomes. They are not completely synthesized during DNA replication and are elongated by specific mechanisms. The structure of the telomeres and the elongation mechanism have not been determined in Dictyostelium discoideum. This organism presents extrachromosomal palindromic elements containing two copies of the rDNA, also present at the end of the chromosomes. In this article the structure of the terminal region of the rDNA is shown to consist of repetitions of the A(G)n sequence where the number of Gs is variable. These repeats extend as a 3’ single stranded region. The G-rich region is preceded by four tandem repetitions of two different DNA motifs. D. discoideum telomere reverse transcriptase homologous protein (TERTHP) presented RNase-sensitive enzymatic activity and was required to maintain telomere structure since terthp-mutant strains presented reorganizations of the DNA terminal regions. These modifications were different in several terthp-mutants and changed with their prolonged culture and subcloning. However, the terthp gene is not essential for D. discoideum proliferation. Telomeres could be maintained in terthp-mutant strains by homologous recombination mechanisms such as ALT (Alternative Lengthening of Telomeres) or HAATI (heterochromatin amplification-mediated and telomerase-independent). In agreement with this hypothesis, the expression of mRNAs coding for several proteins involved in homologous recombination was induced in terthp-mutant strains. Extrachromosomal rDNA could serve as substrate in these DNA homologous recombination reactions. [ABSTRACT FROM AUTHOR]

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 5′ end of env. [ABSTRACT FROM AUTHOR]

The wealth of information deliverable from transcriptome sequencing (RNA-seq) is significant, however current applications for variant detection still remain a challenge due to the complexity of the transcriptome. Given the ability of RNA-seq to reveal active regions of the genome, detection of RNA-seq SNPs can prove valuable in understanding the phenotypic diversity between populations. Thus, we present a novel computational workflow named VAP (Variant Analysis Pipeline) that takes advantage of multiple RNA-seq splice aware aligners to call SNPs in non-human models using RNA-seq data only. We applied VAP to RNA-seq from a highly inbred chicken line and achieved high accuracy when compared with the matching whole genome sequencing (WGS) data. Over 65% of WGS coding variants were identified from RNA-seq. Further, our results discovered SNPs resulting from post transcriptional modifications, such as RNA editing, which may reveal potentially functional variation that would have otherwise been missed in genomic data. Even with the limitation in detecting variants in expressed regions only, our method proves to be a reliable alternative for SNP identification using RNA-seq data. The source code and user manuals are available at . [ABSTRACT FROM AUTHOR]

The diagnosis of Marfan spectrum includes a large number of clinical criteria. Although the identification of pathogenic variants contributes to the diagnostic process, its value to the prediction of clinical outcomes is still limited. An important novelty of the present study is represented by the statistical approach adopted to investigate genotype-phenotype correlation. The analysis has been improved considering the extended genetic information obtained by Next Generation Sequencing (NGS) and combining the effects of both rare and common genetic variants in an inclusive model. To this aim a cohort of 181 patients were analyzed with a NGS panel including 11 genes associated with Marfan spectrum. The genotype-phenotype correlation was also investigated considering the possibility to predict presence of a pathological mutation in Marfan syndrome (MFS) main genes based only on the analysis of phenotypic traits. Results obtained indicate that information about clinical traits can be summarized in a new variable that resulted significantly associated with the probability to find a pathological mutation in MFS main genes. This is important since the choice of the genetic test is often influenced by the phenotypic characterization of patients. Moreover, both rare and common variants were found to significantly contribute to clinical spectrum and their combination allowed to increase the percentage of phenotype variability that could be explained based on genetic factors. Results highlight the opportunity to take advantage of the overall genetic information obtained by NGS data to have a better clinical classification of patients. [ABSTRACT FROM AUTHOR]

Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment. [ABSTRACT FROM AUTHOR]

The human gut microbiota is well established as an important factor in health and disease. Fecal sample microbiota are often analyzed as a proxy for gut microbiota, and characterized with respect to their composition profiles. Modern approaches employ whole genome shotgun next-generation sequencing as the basis for these analyses. Sequencing depth as well as choice of next-generation sequencing data analysis method constitute two main interacting methodological factors for such an approach. In this study, we used 200 million sequence read pairs from one fecal sample for comparing different taxonomy classification methods, using default and custom-made reference databases, at different sequencing depths. A mock community data set with known composition was used for validating the classification methods. Results suggest that sequencing beyond 60 million read pairs does not seem to improve classification. The phylogeny prediction pattern, when using the default databases and the consensus database, appeared to be similar for all three methods. Moreover, these methods predicted rather different species. We conclude that the choice of sequencing depth and classification method has important implications for taxonomy composition prediction. A multi-method-consensus approach for robust gut microbiota NGS analysis is recommended. [ABSTRACT FROM AUTHOR]

There are a few well-known indigenous breeds of Chinese rabbits in Sichuan and Fujian provinces, for which the genetic diversity and population structure have been poorly investigated. In the present study, we successfully employed the restriction-site-associated DNA sequencing (RAD-seq) approach to comprehensively discover genome-wide SNPs of 104 rabbits from four Chinese indigenous breeds: 30 Sichuan White, 34 Tianfu Black, 32 Fujian Yellow and eight Fujian Black. A total of 7,055,440 SNPs were initially obtained, from which 113,973 high-confidence SNPs (read depth ≥ 3, calling rate = 100% and biallelic SNPs) were selected to study the genetic diversity and population structure. The mean polymorphism information content (PIC) and nucleotide diversity (π) of each breed slightly varied with ranging from 0.2000 to 0.2281 and from 0.2678 to 0.2902, respectively. On the whole, Fujian Yellow rabbits showed the highest genetic diversity, which was followed by Tianfu Black and Sichuan White rabbits. The principal component analysis (PCA) revealed that the four breeds were clearly distinguishable. Our results first reveal the genetic differences among these four rabbit breeds in the Sichuan and Fujian provinces and also provide a high-confidence set of genome-wide SNPs for Chinese indigenous rabbits that could be employed for gene linkage and association analyses in the future. [ABSTRACT FROM AUTHOR]

In this study, we describe the development of a PCR-free whole exome sequencing method. Using this method, 2 μg DNA was sufficient for library preparation for whole exome sequencing. Furthermore, the method is simple and makes use of a commercial kit, with additional step of concentrating the captured library by ethanol precipitation. The accuracy of the PCR-free method was found to be equivalent to that of unique molecular identifier-corrected analysis method, which is the commonly used method to detect rare mutations. Thus, the PCR-free whole exome sequencing method is cost-effective as well as efficient in detecting rare mutations. [ABSTRACT FROM AUTHOR]

Plasmodium vivax invasion of human erythrocytes depends on the Duffy Binding Protein (PvDBP) which interacts with the Duffy antigen. PvDBP copy number has been recently shown to vary between P. vivax isolates in Sub-Saharan Africa. However, the extent of PvDBP copy number variation, the type of PvDBP multiplications, as well as its significance across broad samples are still unclear. We determined the prevalence and type of PvDBP duplications, as well as PvDBP copy number variation among 178 Ethiopian P. vivax isolates using a PCR-based diagnostic method, a novel quantitative real-time PCR assay and whole genome sequencing. For the 145 symptomatic samples, PvDBP duplications were detected in 95 isolates, of which 81 had the Cambodian and 14 Malagasy-type PvDBP duplications. PvDBP varied from 1 to >4 copies. Isolates with multiple PvDBP copies were found to be higher in symptomatic than asymptomatic infections. For the 33 asymptomatic samples, PvDBP was detected with two copies in two of the isolates, and both were the Cambodian-type PvDBP duplication. PvDBP copy number in Duffy-negative heterozygotes was not significantly different from that in Duffy-positives, providing no support for the hypothesis that increased copy number is a specific association with Duffy-negativity, although the number of Duffy-negatives was small and further sampling is required to test this association thoroughly. [ABSTRACT FROM AUTHOR]

Micro RNAs (miRNAs) are small single strand non-coding RNAs that regulate gene expression at the post-transcriptional level, either by translational inhibition or mRNA degradation based on the extent of complementarity between the miRNA and its target mRNAs. Potato (Solanum tuberosum L.) is the most important horticultural crop in Argentina. Achieving an integrated control of diseases is crucial for this crop, where frequent agrochemical applications, particularly fungicides, are carried out. A promising strategy is based on promoting induced resistance through the application of environmentally friendly compounds such as phosphites, inorganic salts of phosphorous acid. The use of phosphites in disease control management has proven to be effective. Although the mechanisms underlying their effect remain unclear, we postulated that miRNAs could be involved. Therefore we performed next generation sequencing (NGS) in potato leaves treated and non treated with potassium phosphite (KPhi). We identified 25 miRNAs that were expressed differentially, 14 already annotated in miRBase and 11 mapped to the potato genome as potential new miRNAs. A prediction of miRNA targets showed genes related to pathogen resistance, transcription factors, and oxidative stress. We also analyzed in silico stress and phytohormone responsive cis-acting elements on differentially expressed pre miRNAs. Despite the fact that some of the differentially expressed miRNAs have been already identified, this is to our knowledge the first report identifying miRNAs responsive to a biocompatible stress resistance inducer such as potassium phosphite, in plants. Further characterization of these miRNAs and their target genes might help to elucidate the molecular mechanisms underlying KPhi-induced resistance. [ABSTRACT FROM AUTHOR]

Background: Tyrosine kinase inhibitors targeted to anaplastic lymphoma kinase (ALK) have been demonstrated to be effective for lung cancer patients with an ALK fusion gene. Application of liquid biopsy, i.e., detection and quantitation of the fusion product in plasma cell-free DNA (cfDNA), could improve clinical practice. To detect ALK fusions, because fusion breakpoints occur somewhere in intron 19 of the ALK gene, sequencing of the entire intron is required to locate breakpoints. Results: We constructed a target sequencing system using an adapter and a set of primers that cover the entire ALK intron 19. This system can amplify fragments, including breakpoints, regardless of fusion partners. The data analysis pipeline firstly detected fusions by alignment to selected target sequences, and then quantitated the fusion alleles aligning to the identified breakpoint sequences. Performance was validated using 20 cfDNA samples from ALK-positive non-small cell lung cancer patients and samples from 10 healthy volunteers. Sensitivity and specificity were 50 and 100%, respectively. Conclusions: We demonstrated that PCR-based target sequencing using a tiling primer set and two-step mapping/alignment quantitatively detected ALK fusions in cfDNA from lung cancer patients. The system offers an alternative to existing approaches based on hybridization capture. [ABSTRACT FROM AUTHOR]

Background: More than 11,000 laboratories and companies developed their own next-generation sequencing (NGS) for screening and diagnosis of various diseases including cancer. Although inconsistencies of mutation calls as high as 43% in databases such as GDSC (Genomics of Drug Sensitivity in Cancer) and CCLE (Cancer Cell Line Encyclopedia) have been reported, not many studies on the reasons for the inconsistencies have been published. Methods: Targeted-NGS analysis of 151 genes in 35 cell lines common to GDSC and CCLE was performed, and the results were compared with those from GDSC and CCLE wherein whole-exome- or highly-multiplex NGS were employed. Results: In the comparison, GDSC and CCLE had a high rate (40–45%) of false-negative (FN) errors which would lead to high rate of inconsistent mutation calls, suggesting that highly-multiplex NGS may have high rate of FN errors. We also posited the possibility that targeted NGS, especially for the detection of low-level cancer cells in cancer tissues might suffer significant FN errors. Conclusion: FN errors may be the most important errors in NGS testing for cancer; their evaluation in laboratory-developed NGS tests is needed. [ABSTRACT FROM AUTHOR]

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines, organic cations that are implicated in many cellular processes. The enzyme is regulated at the post-translational level by an unusual system that includes antizymes (AZs) and antizyme inhibitors (AZINs). Most studies on this complex regulatory mechanism have been focused on human and rodent cells, showing that AZINs (AZIN1 and AZIN2) are homologues of ODC but devoid of enzymatic activity. Little is known about Xenopus ODC and its paralogues, in spite of the relevance of Xenopus as a model organism for biomedical research. We have used the information existing in different genomic databases to compare the functional properties of the amphibian ODC1, AZIN1 and AZIN2/ODC2, by means of transient transfection experiments of HEK293T cells. Whereas the properties of xlODC1 and xlAZIN1 were similar to those reported for their mammalian orthologues, the former catalyzing the decarboxylation of L-ornithine preferentially to that of L-lysine, xlAZIN2/xlODC2 showed important differences with respect to human and mouse AZIN2. xlAZIN2 did not behave as an antizyme inhibitor, but it rather acts as an authentic decarboxylase forming cadaverine, due to its higher affinity to L-lysine than to L-ornithine as substrate; so, in accordance with this, it should be named as lysine decarboxylase (LDC) or lysine/ornithine decarboxylase (LODC). In addition, AZ1 stimulated the degradation of xlAZIN2 by the proteasome, but the removal of the 21 amino acid C-terminal tail, with a sequence quite different to that of mouse or human ODC, made the protein resistant to degradation. Collectively, our results indicate that in Xenopus there is only one antizyme inhibitor (xlAZIN1) and two decarboxylases, xlODC1 and xlLDC, with clear preferences for L-ornithine and L-lysine, respectively. [ABSTRACT FROM AUTHOR]

The five-year survival rate of patients diagnosed with advanced pancreatic ductal adenocarcinoma (PDAC) has remained static at <5% despite decades of research. With the exception of erlotinib, clinical trials have failed to demonstrate the benefit of any targeted therapy for PDAC despite promising results in preclinical animal studies. The development of more refined mouse models of PDAC which recapitulate the carcinogenic progression from non-neoplastic, adult exocrine subsets of pancreatic cells to invasive carcinoma in humans are needed to facilitate the accurate translation of therapies to the clinic. To study acinar cell-derived PDAC initiation, we developed a genetically engineered mouse model of PDAC, called KPT, utilizing a tamoxifen-inducible Cre recombinase/estrogen receptor (ESR1) fusion protein knocked into the Ptf1a locus to activate the expression of oncogenic KrasG12D and Trp53R270H alleles in mature pancreatic acinar cells. Oncogene-expressing acinar cells underwent acinar-to-ductal metaplasia, and formed pancreatic intraepithelial neoplasia lesions following the induction of oncogene expression. After a defined latency period, oncogene-expressing acinar cells initiated the formation of highly differentiated and fibrotic tumors, which metastasized to the lungs and liver. Whole-transcriptome analysis of microdissected regions of acinar-to-ductal metaplasia and histological validation experiments demonstrated that regions of acinar-to-ductal metaplasia are characterized by the deposition of the extracellular matrix component hyaluronan. These results indicate that acinar cells expressing KrasG12D and Trp53R270H can initiate PDAC development in young adult mice and implicate hyaluronan deposition in the formation of the earliest characterized PDAC precursor lesions (and the progression of pancreatic cancer). Further studies are necessary to provide a comprehensive characterization of PDAC progression and treatment response in KPT mice and to investigate whether the KPT model could be used as a tool to study translational aspects of acinar cell-derived PDAC tumorigenesis. [ABSTRACT FROM AUTHOR]

Dendrograms are a way to represent relationships between organisms. Nowadays, these are inferred based on the comparison of genes or protein sequences by taking into account their differences and similarities. The genetic material of choice for the sequence alignments (all the genes or sets of genes) results in distinct inferred dendrograms. In this work, we evaluate differences between dendrograms reconstructed with different methodologies and for different sets of organisms chosen at random from a much larger set. A statistical analysis is performed to estimate fluctuations between the results obtained from the different methodologies that allows us to validate a systematic approach, based on the comparison of the organisms’ metabolic networks for inferring dendrograms. This has the advantage that it allows the comparison of organisms very far away in the evolutionary tree even if they have no known ortholog gene in common. Our results show that dendrograms built using information from metabolic networks are similar to the standard sequence-based dendrograms and can be a complement to them. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) expressed on the surface of Gram-negative bacteria activates pro-inflammatory pathways, dys-regulates the function of endometrial cells and is a key player in the mechanisms involved in endometritis. This study aimed to investigate the effects of LPS on bovine endometrial epithelial cells (bEEC) from whole transcriptome with a special focus on genes involved in embryo-maternal interactions. Following in vitro culture, bEEC from three cows were exposed to 0, 2, and 8 μg/mL LPS for 24h. RNA samples extracted at 0 and 24 hours were analyzed by RNA sequencing (RNA-seq). At 24h, 2035 differentially expressed genes (DEGs) were identified between controls and samples treated with 2 μg/mL LPS. Gene ontology analysis showed that over-expressed DEGs were associated to immune response, response to stress and external stimuli, catalytic activity, and cell cycle. Genes associated with cell membrane and cell adhesion pathways were under-expressed. LPS induced changes in expression of specific genes related to embryo-maternal interactions including under-expression of eight members of the cadherin superfamily, over-expression of six members of the mucin family, and differential expression of a large set of genes binding the above molecules and of more than 20 transcripts coding for cytokines and their receptors. Type I interferon-τ dependent genes were also over-expressed. From a sub-set of 19 genes, (biological replicates of bEEC from cows taken at time 6 (n = 3), 24 (n = 6) and 48 hours (n = 3), and 2 technical replicates per sample) differential gene expression was confirmed by RT2-qPCR (r2 between fold changes at 24 hours by RT2-qPCR and RNA-seq = 0.97). These results indicate that LPS affects the function of bEEC in many ways by differential transcription, glycolytic metabolism and oxidative stress. Many transcriptomic signatures related to implantation and embryo maternal interactions were strongly affected by LPS. These results pave the way for further studies to investigate the duration of these changes and their possible impact on endometrial function and fertility. [ABSTRACT FROM AUTHOR]

The ribosome moves between distinct structural states and is organized into multiple functional domains. Here, we examined hundreds of occurrences of pairwise through-space communication between nucleotides in the ribosome small subunit RNA using RNA interaction groups analyzed by mutational profiling (RING-MaP) single-molecule correlated chemical probing in bacterial cells. RING-MaP revealed four structural communities in the small subunit RNA, each distinct from the organization defined by the RNA secondary structure. The head domain contains 2 structural communities: the outer-head contains the pivot for head swiveling, and an inner-head community is structurally integrated with helix 44 and spans the entire ribosome intersubunit interface. In-cell binding by the antibiotic spectinomycin (Spc) barely perturbs its local binding pocket as revealed by the per-nucleotide chemical probing signal. In contrast, Spc binding overstabilizes long-range RNA–RNA contacts that extend 95 Å across the ribosome that connect the pivot for head swiveling with the axis of intersubunit rotation. The two major motions of the small subunit—head swiveling and intersubunit rotation—are thus coordinated via long-range RNA structural communication, which is specifically modulated by Spc. Single-molecule correlated chemical probing reveals trans-domain structural communication and rationalizes the profound functional effects of binding by a low–molecular-mass antibiotic to the megadalton ribosome. [ABSTRACT FROM AUTHOR]

Increased use of salting to de-ice roadways, especially in urban areas, is leading to elevated salinity levels in soil as well as surface- and ground water. This salt pollution may cause long-term ecological changes to soil and aquatic microbial communities. In this study, we examined the impact on microbial communities in soils exposed to urban road salt runoff using both culturing and 16S amplicon sequencing. Both methods showed an increase in halophilic Bacteria and Archaea in samples from road salt-exposed areas and suggested that halophiles are becoming persistent members of microbial communities in urban, road salt-impacted soils. Since salt is a pollutant that can accumulate in soils over time, it is critical to begin assessing its impact on the environment immediately. Toward this goal, we have developed a facile semi-quantitative assay utilizing halophilic microbes as biomarkers to evaluate on-going salt pollution of soils. [ABSTRACT FROM AUTHOR]

We investigated the feasibility of using next-generation sequencing (NGS) technique using molecular barcoding technology to detect MYD88 L265P mutation in unselected peripheral blood mononuclear cells (PBMCs) in 52 patients with Waldenström’s macroglobulinemia [1] and 21 patients with IgM-monoclonal gammopathy of undetermined significance (MGUS). The NGS technique successfully detected the MYD88 L265P in unselected PBMCs at a sensitivity of 0.02%, which was ×5 higher than that of AS-PCR. All the results between paired BM and PB samples from 2 IgM MGUS and 4 untreated WM patients matched completely. MYD88 L265P mutation was detected in 14/21 (66.7%), 14/19 (73.7%), and 10/33 (30.3%) with the median mutant allele burden of 0.36% (range, 0.06–2.85%), 0.48% (range, 0.02–32.3%), and 0.16% (range, 0.02–33.8%), in IgM-MGUS, untreated WM, and previously treated WM, respectively. Multiple linear regression analysis identified an absolute peripheral lymphocyte count as the positive predictor of PB mutant allele burden (R2 = 0,72, P<0.0001). Our non-invasive, simple NGS method has the potential to detect MYD88 L265P mutations in PBMCs of IgM MGUS and WM patients, which may especially utilized for monitoring minimal residual tumor burden after treatment. [ABSTRACT FROM AUTHOR]

Scrub typhus has been documented since 1932 in Vietnam, however, the disease burden of scrub typhus remains poorly understood in the country. We conducted this study to describe the phylogenetic analysis of the 56-kDa type-specific antigen (TSA) gene of Orientia tsutsugamushi associated with PCR positive cases of scrub typhus. Of 116 positive samples, 65 type-specific antigen gene sequences were obtained and classified into 3 genogroups: Karp, Kato and Gilliam. The Karp genogroup was the most frequently detected phylogenetic cluster in the study with 30 samples (46%), followed by Kato and Gilliam with 20 (31%) and 15 (23%), respectively. All sequences showed 94–100% nucleotide similarity to reference sequences collected in the central part of Vietnam in 2017. Patients infected with Karp genogroup were more likely to have significant thrombocytopenia than the other genogroups. These results suggest that any scrub typhus vaccine considered for use in Vietnam should provide protection against each of these 3 genogroups. [ABSTRACT FROM AUTHOR]

Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for avooxin from P. enovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth β-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2. [ABSTRACT FROM AUTHOR]

Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for avooxin from P. enovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth β-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2. [ABSTRACT FROM AUTHOR]

Background: Genomic data have become major resources to understand complex mechanisms at fine-scale temporal and spatial resolution in functional and evolutionary genetic studies, including human diseases, such as cancers. Recently, a large number of whole genomes of evolving populations of yeast (Saccharomyces cerevisiae W303 strain) were sequenced in a time-dependent manner to identify temporal evolutionary patterns. For this type of study, a chromosome-level sequence assembly of the strain or population at time zero is required to compare with the genomes derived later. However, there is no fully automated computational approach in experimental evolution studies to establish the chromosome-level genome assembly using unique features of sequencing data. Methods and results: In this study, we developed a new software pipeline, the integrative meta-assembly pipeline (IMAP), to build chromosome-level genome sequence assemblies by generating and combining multiple initial assemblies using three de novo assemblers from short-read sequencing data. We significantly improved the continuity and accuracy of the genome assembly using a large collection of sequencing data and hybrid assembly approaches. We validated our pipeline by generating chromosome-level assemblies of yeast strains W303 and SK1, and compared our results with assemblies built using long-read sequencing and various assembly evaluation metrics. We also constructed chromosome-level sequence assemblies of S. cerevisiae strain Sigma1278b, and three commonly used fungal strains: Aspergillus nidulans A713, Neurospora crassa 73, and Thielavia terrestris CBS 492.74, for which long-read sequencing data are not yet available. Finally, we examined the effect of IMAP parameters, such as reference and resolution, on the quality of the final assembly of the yeast strains W303 and SK1. Conclusions: We developed a cost-effective pipeline to generate chromosome-level sequence assemblies using only short-read sequencing data. Our pipeline combines the strengths of reference-guided and meta-assembly approaches. Our pipeline is available online at including a Docker image, as well as a Perl script, to help users install the IMAP package, including several prerequisite programs. Users can use IMAP to easily build the chromosome-level assembly for the genome of their interest. [ABSTRACT FROM AUTHOR]

DNA methylation has frequently been implicated in sex determination and differentiation in teleost species. In order to detect the DNA methylation patterns established during sexual differentiation in tiger pufferfish T. rubripes, we performed comprehensive whole genome methylation sequencing and analyses of the gonads of male, female, and pseudo male. We obtained a total of 33.12, 32.44, and 31.60 Gb clean data for male, female, and pseudo male, with a sequencing depth of 66.44×, 60.47× and 54.86×, respectively. The methylation level of cytosine (C) residues in the genomic DNA from gonads was 11.016%, 10.428%, and 11.083% in male, female, and pseudo male, respectively. More than 65% of C methylation was at CpG sites, and less than 1% was at CHG and CHH sites. In each regulatory element, there were low methylation levels on both sides of the transcription start site, and higher methylation levels in exons, introns, and downstream of genes. The highest mCpG was on chromosome 8 and the lowest mCpG was on chromosome 5. Comparisons of whole-genome DNA methylation between pairs of samples revealed that there were 3,173 differentially methylated regions (DMRs) between female and male, and 3,037 DMRs between male and pseudo male, corresponding to 0.232% and 0.223% of the length of the genome, respectively. There were only 1,635 DMRs between female and pseudo male, representing 0.127% of the length of the genome. A number of differentially methylated genes (DMGs) related to sex determination and differentiation were selected, such as amhr2 and pfcyp19a. After Bisulfite Sequencing PCR (BSP) verification, amhr2 was exhibited low methylation level in normal males and pseudo male, and high methylation level in normal females but pfcyp19a showed low methylation level in normal females and high methylation level in normal males and pseudo males. These results provide information about the molecular epigenetic mechanisms of DNA methylation during low-temperature induced masculinization of tiger pufferfish, and increase our understanding of the mechanisms of sex determination and differentiation in this important aquaculture fish species. [ABSTRACT FROM AUTHOR]

Understanding intrinsic and acquired resistance is crucial to overcoming cancer chemotherapy failure. While it is well-established that intratumor, subclonal genetic and phenotypic heterogeneity significantly contribute to resistance, it is not fully understood how tumor sub-clones interact with each other to withstand therapy pressure. Here, we report a previously unrecognized behavior in heterogeneous tumors: cooperative adaptation to therapy (CAT), in which cancer cells induce co-resistant phenotypes in neighboring cancer cells when exposed to cancer therapy. Using a CRISPR/Cas9 toolkit we engineered phenotypically diverse non-small cell lung cancer (NSCLC) cells by conferring mutations in Dicer1, a type III cytoplasmic endoribonuclease involved in small non-coding RNA genesis. We monitored three-dimensional growth dynamics of fluorescently-labeled mutant and/or wild-type cells individually or in co-culture using a substrate-free NanoCulture system under unstimulated or drug pressure conditions. By integrating mathematical modeling with flow cytometry, we characterized the growth patterns of mono- and co-cultures using a mathematical model of intra- and interspecies competition. Leveraging the flow cytometry data, we estimated the model’s parameters to reveal that the combination of WT and mutants in co-cultures allowed for beneficial growth in previously drug sensitive cells despite drug pressure via induction of cell state transitions described by a cooperative game theoretic change in the fitness values. Finally, we used an ex vivo human tumor model that predicts clinical response through drug sensitivity analyses and determined that cellular and morphologic heterogeneity correlates to prognostic failure of multiple clinically-approved and off-label drugs in individual NSCLC patient samples. Together, these findings present a new paradox in drug resistance implicating non-genetic cooperation among tumor cells to thwart drug pressure, suggesting that profiling for druggable targets (i.e. mutations) alone may be insufficient to assign effective therapy. [ABSTRACT FROM AUTHOR]

The anticariogenic activity of the extract of Glycyrrhiza uralensis (licorice) has been well documented. We recently developed an herbal lollipop containing licorice extracts with Glycyrrhizol A, the compound displaying strong antimicrobial activity against Streptococcus mutans. Preliminary testing showed that the herbal lollipop reduced salivary S. mutans counts in vivo. In this study, we aimed to further test the efficacy of this herbal lollipop for reducing salivary S. mutans levels, and investigate its impact on salivary microbiome. Using a well-established in vitro oral microbiome model, we showed that licorice extract displays targeted killing against S. mutans without affecting the biodiversity of the community. In vivo study corroborated in vitro findings, showing for high caries-risk children aged 3–6 with salivary S. mutans levels >5x105 cells/ml, daily use of 2 licorice-containing lollipops for 3 weeks significantly reduced salivary S. mutans levels compared to the control group. Salivary microbiome analysis showed either no change or even increase in phylogenetic diversity of the oral community following herbal lollipop usage. Although further study with longer term observation is needed, these results suggest that use of licorice extract-containing lollipops can be as a simple and effective way to reduce the risk of dental caries in children. [ABSTRACT FROM AUTHOR]

The analyses of large volumes of metagenomic data extracted from aggregate populations of microscopic organisms residing on and in the human body are advancing contemporary understandings of the integrated participation of microbes in human health and disease. Next generation sequencing technology facilitates said analyses in terms of diversity, community composition, and differential abundance by filtering and binning microbial 16S rRNA genes extracted from human tissues into operational taxonomic units. However, current statistical tools restrict study designs to investigations of limited numbers of host characteristics mediated by limited numbers of samples potentially yielding a loss of relevant information. This paper presents a Bayesian hierarchical negative binomial model as an efficient technique capable of compensating for multivariable sets including tens or hundreds of host characteristics as covariates further expanding analyses of human microbiome count data. Simulation studies reveal that the Bayesian hierarchical negative binomial model provides a desirable strategy by often outperforming three competing negative binomial model in terms of type I error while simultaneously maintaining consistent power. An application of the Bayesian hierarchical negative binomial model using subsets of the open data published by the American Gut Project demonstrates an ability to identify operational taxonomic units significantly differentiable among persons diagnosed by a medical professional with either inflammatory bowel disease or irritable bowel syndrome that are consistent with contemporary gastrointestinal literature. [ABSTRACT FROM AUTHOR]

Rapid improvements in DNA sequencing technology have resulted in long genome sequences for a large number of similar isolates with a wide range of single nucleotide polymorphism (SNP) rates, where some isolates can have thousands of times lower SNP rates than others. Genome sequences of this kind are a challenge to existing methods for construction of phylogenetic trees. We address the issues by developing a hierarchical approach to phylogeny construction. In this method, the construction is performed at multiple levels, where at each level, groups of isolates with similar levels of similarity are identified and their phylogenetic trees are constructed. Time savings are achieved by using a sufficiently large number of columns from the input alignment, instead of all its columns. Our results show that the new approach is 20-60 times more efficient than existing programs and more accurate in situations where highly similar isolates have a wide range of SNP rates. [ABSTRACT FROM AUTHOR]