Infectious bronchitis virus (IBV) is a highly variable RNA virus that affects chickens worldwide. Due to its inherited tendency to suffer point mutations and recombination events during viral replication, emergent IBV strains have been linked to nephropathogenic and reproductive disease that are more severe than the typical respiratory disease, leading, in some cases, to mortality, severe production losses, and/or unsuccessful vaccination. QX and DMV/1639 strains are examples of the above-mentioned IBV evolutionary pathway and clinical outcome. In this study, our purpose was to systematically compare whole genomes of QX and DMV strains looking at each IBV gene individually. Phylogenetic analyses and amino acid site searches were performed in datasets obtained from GenBank accounting for all IBV genes and using our own relevant sequences as a basis. The QX dataset studied is more genetically diverse than the DMV dataset, partially due to the greater epidemiological diversity within the five QX strains used as a basis compared to the four DMV strains from our study. Historically, QX strains have emerged and spread earlier than DMV strains in Europe and Asia. Consequently, there are more QX sequences deposited in GenBank than DMV strains, assisting in the identification of a larger pool of QX strains. It is likely that a similar evolutionary pattern will be observed among DMV strains as they develop and spread in North America.

During the last decades West Nile Virus (WNV) outbreaks have continuously occurred in the Mediterranean area. In August 2020 a new WNV outbreak affected 71 people with meningoencephalitis in Andalusia and 6 more cases in Extremadura (south-west of Spain), causing a total of eight deaths. The whole genomes of four viral isolates were obtained and phylogenetically analyzed in the context of recent outbreaks. The Andalusian viral samples belonged to the lineage 1 and were relatively similar to previous outbreaks occurred in the Mediterranean region. Here we present a detailed analysis of the outbreak, including an extensive phylogenetic study.

We presented here the first draft genome sequence of the trypanosomatid Herpetomonas muscarum ingenoplastis. This parasite was isolated repeatedly in the black blowfly, Phormia regina. This is the first draft genome of a flagellate from the phylogenetically distinct clade of Trypanosomatidae.

Appropriate vaccine selection is crucial in the control of foot-and-mouth disease (FMD). Vaccination can prevent clinical disease and reduces viral shedding, but there is a lack of cross-protection between the seven serotypes and their sublineages, making the selection of an adequately protective vaccine difficult. Since the exact composition of their vaccines is not consistently disclosed by all manufacturers, incompatibility of the strains used for vaccination with regionally circulating strains can cause vaccination campaigns to fail. Here, we present a deep sequencing approach for polyvalent inactivated FMD vaccines that can identify all component strains by their genome sequences. The genomes of all strains of a commercial pentavalent FMD vaccine were de-novo assembled and the vaccine composition determined semi-quantitatively. The genome assembly required high stringency parameters to prevent misassemblies caused by conserved regions of the genome shared by related strains. In contrast, reference-guided assembly is only recommended in cases where the number of strains is previously known and appropriate reference sequences are available. The presented approach can be applied not only to any inactivated whole-virus FMD vaccine, but also to vaccine quality testing in general and allows for better decision-making for vaccines with unknown composition.

Astroviruses (AstVs) are occurs globally and are common causes of gastroenteritis in human and animals. The genetic diversity and epidemiology of AstVs in Africa is not well known, hence, we aimed to genetically characterize astroviruses in asymptomatic smallholder piglets in East Africa. Twenty-four samples randomly selected from 446 piglets (<6 months old), initially collected for rotavirus study, was sequenced for metagenomic analysis. Thirteen (13/24) samples had contigs with high identity to genus Mamastrovirus. Analysis of 7 strains with complete (or near complete) genome revealed variable nucleotide and amino acid sequence identities with known PoAstV strains. The U083 and K321 strains had nucleotide sequence similarities ranging from 66.4 to 75.4 % to the known PoAstV2 strains, nucleotide sequence similarity of U460 strain with known PoAstV3 ranged 57.0 to 65.1 % to the, while K062, K366, K451, and K456 strains showed nucleotide sequence similarities of 63.5 to 80 % to the known PoAstV4 strains. The low sequence identities (<90 %) indicate that novel genotypes of PoAstVs are circulating in the study area. Multiple recombination events were detected in our PoAstV4 strains, indicating that the genetic diversity observed in these strains may be due to recombination. Importantly, we identified potential candidate epitopes with conserved peptides in our PoAstV strains that could aid in the design of immune diagnosis tools and subunit vaccines. Our data provide new intuitions into the genetic structure of porcine astroviruses in East African.

Human metapneumovirus (HMPV) is an important cause of upper and lower respiratory tract disease in individuals of all ages. It is estimated that most individuals will be infected by HMPV by the age of 5 years old. Despite this burden of disease, there remains caveats in our knowledge of virus global genetic diversity due to a lack of HMPV sequencing, particularly at whole genome scale. The purpose of this study was to create a simple and robust approach for HMPV whole genome sequencing to be used for genomic epidemiological studies. To design our assay, all available HMPV full length genome sequences were downloaded from the NCBI GenBank database and used to design four primer sets to amplify long, overlapping amplicons spanning the viral genome and, importantly, specific to all known HMPV subtypes. These amplicons were then pooled and sequenced on an Illumina iSeq; however the approach is suitable to other common NGS platforms. We demonstrate the utility of this method using a representative subset of clinical samples and examine these sequences using a phylogenetic approach. Here we present an amplicon-based method for the whole genome sequencing of HMPV from clinical extracts that can be used to better inform genomic studies of HMPV epidemiology and evolution.

Triploidy in cancer is associated with poor prognosis but its origins remain unclear. Here, we attempted to differentiate between random chromosomal and whole-genome origins of cancer triploidy. In silico meta-analysis was performed on 15 male malignant and 5 benign tumour cohorts (2928 karyotypes) extracted from the Mitelman Database, comparing their ploidy and combinations of sex chromosomes. A distinct near-triploid fraction was observed in all malignant tumour types, being especially high in seminoma. For all tumour types, X-chromosome doubling, predominantly observed as XXY, correlated strongly with the near-triploid state (r≈0.9, p<0.001), negatively correlated with near-diploidy, and did not correlate with near-tetraploidy. A smaller near-triploid component with a doubled X-chromosome was also present in 3 of 5 benign tumour types, especially notable in colon adenoma. Principal Component Analysis revealed a non-random correlation structure shaping the X-chromosome disomy distribution across all tumour types. We suggest that doubling of the maternal genome followed by pedogamic fusion with a paternal genome (a possible mimic of the fertilization aberration, 69, XXY digyny) associated with meiotic reprogramming may be responsible for the observed rearrangements of genome complements leading to cancer triploidy. The relatively frequent loss of the Y-chromosome results secondary from chromosome instability.

Whole-genome sequencing has advanced our understanding of the population structure of the pathogenic species complex Cryptococcus gattii, which has allowed for the phylogenomic specification of previously described major molecular type groupings and novel lineages. Recently, isolates collected in Mexico in the 1960s were determined to be genetically distant from other known molecular types and were classified as VGVI. We sequenced four clinical isolates and one veterinary isolate collected in the southwestern U.S. and Argentina during 2012-2021. Phylogenomic analysis groups these genomes with those of the Mexican VGVI isolates, expanding VGVI into a clade and establishing this molecular type as a clinically important population. These findings also potentially expand the known Cryptococcus ecological range with a previously unrecognized endemic area.

Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25-1 G>T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48-12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which in a human colonic cancer cell line controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

Colorectal cancer (CRC) shows one of the largest proportions of familial cases among different malignancies, but only 5-10% of all CRC cases are linked to mutations in established predisposition genes. Thus, familial CRC constitutes a promising target for the identification of novel, high- to moderate-penetrance germline variants underlying cancer susceptibility by next generation sequencing. In this study, we performed whole genome sequencing on 3 members of a family with CRC aggregation. Subsequent integrative in silico analysis using our in-house developed variant prioritization pipeline resulted in the identification of a novel germline missense variant in SRC gene (V177M), a proto-oncogene highly upregulated in CRC. Functional validation experiments in HT-29 cells showed that introduction of SRCV177M resulted in increased cell proliferation and enhanced protein expression of phospho-SRC (Y419), a potential marker for SRC activity. Upregulation of paxillin, β-Catenin and STAT3 mRNA levels, increased levels of phospho-ERK, CREB and CCND1 proteins and downregulation of the tumor suppressor p53 further proposed the activation of several pathways due to the SRCV177M variant. The findings of our pedigree-based study contribute to the exploration of the genetic background of familial CRC and bring insights into the molecular basis of upregulated SRC activity and downstream pathways in colorectal carcinogenesis.

Klebsiella pneumoniae (Kp) has gained prominence in the last two decades due to its global spread as a multi-drug resistant (MDR) pathogen. Further, Carbapenem-Resistant Kp are emerging at an alarming rate. The objective of this study was (1) to evaluate the prevalence of β-lactamases, especially carbapenemases in Kp isolates from India, (2) determine the most prevalent sequence type (ST) & plasmids, and their association with β-lactamases. Clinical samples of K. pneumoniae (n=65) were collected from various pathology lab, drug susceptibility and minimum inhibitory concentrations (MIC) were detected. Whole genome sequencing (WGS) was done for (n=22) resistant isolates and WGS analysis was performed using various bioinformatics tools. Additional Indian MDR Kp genomes (n=187) were retrieved using Pathosystems Resource Integration Center (PATRIC) database. Detection of β-lactamase genes, location, plasmid replicons, and ST type of genomes were carried out using CARD, mlplasmids, PlasmidFinder, and PubMLST respectively. All data were analyzed and summarized using iTOL tool. ST231 was highest, followed by ST147, ST2096 & ST14 among Indian isolates. blaAmpH was detected as the most prevalent gene followed by blaCTX-M-15, blaTEM-1. Among carbapenemase genes, blaOXA-232 was prevalent and associated with ST231, ST2096 and ST14, which was followed by blaNDM-5 which was observed to prevalent in ST147, ST395 &ST437. ST231 genomes were most commonly found to carry Col440I and ColKP3 plasmids. ST16 carried mainly ColKP3, and Col (BS512) was abundantly present in ST147 genomes. One Kp isolate with novel MLST profile was identified, which carried blaCTX-M-15, blaOXA-1 and blaTEM-1. ST16 &ST14 from this study, which is mostly dual producer of carbapenem and ESBL genes, could be emerging high-risk clones in India.

Whole genome pooled sequence data of 12 Pakistani Teddy goats is analyzed for positive selection signatures as their breed defining characteristics. Selection imprints left in the Teddy genome are unveiled by genomic differentiation after the successful paired-end alignment of 635,357,043 reads with (ARS1) reference genome assembly. Pooled-heterozygosity ( ) and Tajima’s D (TD) are applied for validation and getting better hits of selection signals, while pairwise F_ST statistics is conducted on Teddy vs. Bezoar (wild goat ancestor) for genomic differentiation. Annotation of regions under positive selection reveals 59 genes underlying production and adaptive traits. score ≥ 5 detected six windows having highest scores on Chr. 29, 9, 25, 15 and 14 that harbor HRASLS5, LACE1 and AXIN1 genes which are candidate for embryonic development, lactation and body height. Secondly, TD value of ≤ -2.2 showed 4 windows with very strong hits on Chr.5 & 9 harbor STIM1 and ADM genes related to body mass and weight. Lastly, F_ST analysis generated three strong signals with threshold ≤ 0.42 on Chr.12 & 5 harbor ITGB1 gene associated with milk production & lactation traits. Other significant selection signatures encompass genes associated with wool production, prolificacy, immunity and coat colors. In brief, this study identified the genes under selection in this Pakistani goat breed that will be helpful to refining future breeding policies and converging required productive traits within and across other goat breeds and to explore full genetic potential of this valued livestock species.

Chickens have extensive phenotypic variation. The Wuhua yellow chicken (WHYC) is an important traditional yellow-feathered chicken in China, characterized by white tail feathers, white flight feathers, and strong disease resistance. However, the genomic basis of traits associated with WHYC is still poorly understood. In this study, whole genome resequencing was performed with an average coverage of 20.77-fold to investigate heritable variation and identify selection signals in WHYC. Reads were mapped onto the chicken reference genome (Galgal5) with a coverage of 85.95%. After quality control, 11,953,471 SNPs and 1,069,574 InDels were obtained. In addition, 41,408 structural variants and 33,278 copy number variants were found. A comparative genomic analysis of WHYC and other yellow-feathered chicken showed that selected regions were enriched in genes involved in transport and catabolism, immune system, infectious diseases, signal transduction, and signaling molecules and interaction. Several genes associated with disease resistance were identified, including IFNA, IFNB, CD86, IL18, IL11RA, VEGFC, and ATG10. Furthermore, PMEL and TYRP1 may contribute to the coloring of white feathers in WHYC. These findings improve our understanding of the genetic characteristics of WHYC and may contribute to future breed improvement.

Beta-lactam resistant bacteria, commonly resident in tertiary hospitals, have emerged as a worldwide health problem because of ready-to-eat vegetable intake. We aimed to characterize the genes providing resistance to beta-lactam antibiotics in Enterobacteriaceae, isolated from five commercial salad brands for human consumption in Mexico City. 25 samples were collected, grow in blood agar plates, the bacteria were biochemistry identified and antimicrobial susceptibility testing was done, the carried family genes were identified by endpoint PCR and the specific genes were confirmed with WGS by NGS. 12 positive cultures were identified and their microbiological distribution was as follows, 8.3% for Enterobacter aerogene (n=1), 8.3% for Serratia fonricola (n=1), 16.7% for Serratia marcesens (n=2), 16.7% for Klebsiella pneumoniae (n=2), and 50% (n=6) for Enterobacter cloacae. The endpoint PCR results showed 11 colonies positive for blaBIL (91.7%), 11 for blaSHV (91.7%), 11 for blaCTX (97.7%), 12 for blaDHA (100%),4 for blaVIM (33.3%), 2 for blaOXA (16.7%), 2 for blaIMP (16.7%), 1 for blaKPC (8.3%) and 1 for blaTEM (8.3%) gene, all samples were negative blaROB, blaCMY, blaP, blaCFX and blaLAP gene. The sequencing analysis revels a specific genotypes for Enterobacter cloacae (blaSHV-12, blaCTX-M-15, blaDHA-1, blaKPC-2); Serratia marcescens (blaSHV-1, blaCTX-M-3, blaDHA-1, blaVIM-2); Klebsiella pneumoniae (blaSHV-12, blaCTX-M-15, blaDHA-1); Serratia fonticola (blaSHV-12, blaVIM-1, blaDHA-1) and Enterobacter aerogene (blaSHV-1, blaCTX-M-1, blaDHA-1, blaVIM-2, blaOXA-9). Our results indicate that beta-lactam resistant bacteria have acquired integrons with a different number of genes that providing panresistance to beta-lactam antibiotics, including penicillins, oxacillins, cefalosporins, monobactams, carbapenems and imipenems.

Chromosomal rearrangements in N. gonorrhoeae and N. meningitidis were studied with the determination of mobile elements and their role in rearrangements. The results of whole-genome sequencing and de novo genome assembly for 50 N. gonorrhoeae isolates collected in Russia were compared with 96 genomes of N. gonorrhoeae and 138 genomes of N. meningitidis from the databases. Rearrangement events with the determination of the coordinates of syntenic blocks were analyzed using the SibeliaZ software, the minimum number of events that allow one genome to pass into another was calculated using the DCJ-indel model using the UniMoG program. Population-level analysis revealed a stronger correlation between changes in the gene order and phylogenetic proximity for N. meningitidis in contrast to N. gonorrhoeae. Mobile elements were identified, including Correa elements, Spencer-Smith elements (in N. gonorrhoeae), Neisserial intergenic mosaic elements, IS elements of IS5, IS30, IS110, IS1595 groups, Nf1-Nf3 prophages, NgoФ1-NgoФ9 prophages, Mu-like prophages Pnm1, Pnm2, MuMenB (in N. meningitidis). More than 44% of the observed rearrangements most likely occurred with the participation of mobile elements, including prophages. No differences were found between the Russian and global N. gonorrhoeae population both in terms of rearrangement events and in the number of transposable elements in genomes.

Komagataeibacter spp. have been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose producing bacteria capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in minimal medium containing glucose (3.3±0.3 g/L), pure glycerol (2.2±0.1 g/L) and crude glycerol (2.1±0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in minimal medium supplemented with acetate, BC production was not observed. However, in presence of readily utilizable substrate, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.

Abstract: Infectious bronchitis virus (IBV) induces respiratory and urogenital disease in chickens. Although IBV replicates in the gastrointestinal tract, enteric lesions are uncommon. We have reported a case of runting-stunting syndrome in commercial broilers from which an IBV variant was isolated from the intestines. The isolate, CalEnt, demonstrated an enteric tissue tropism in chicken embryos and SPF chickens experimentally. Here, we determined the full genome of CalEnt and compared it to other IBV strains, in addition to comparing the pathobiology of CalEnt and M41 in commercial broilers. Despite the high whole-genome identity to other IBV strains, CalEnt is rather unique in nucleotide composition. The S gene phylogenetic analyses showed great similarity between CalEnt and Cal 99. Clinically, vent staining was slightly more frequent in CalEnt-infected birds than those challenged with M41. Furthermore, IBV IHC detection was more evident and the viral shedding in feces was overall higher with the CalEnt challenge compared with M41. Despite underlying intestinal lesions caused by coccidiosis and salmonellosis vaccination, microscopic lesions in CalEnt-infected chickens were more severe than in M41-infected chickens or controls, supporting the enteric tropism of CalEnt. Further studies in SPF chickens are needed to determine the pathogenesis of the virus, its molecular mechanisms for the enteric tropism, and its influence in intestinal health.

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

Whole-genome DNA sequencing of Lactobacillus D75 and D76 strains (Vitaflor, Russia) was performed using the PacBio RS II platform, followed by de novo assembly with SMRT Portal 2.3.0. The average nucleotide identity (ANI) test showed that both strains belong to the Lactobacillus helveticus, but not the L. acidophilus as previously assumed. 31 exopolysaccharide (EPS) production genes (nine of which form a single genetic cluster), 13 adhesion genes, 38 milk protein and 11 milk sugar utilization genes, 13 genes for and against specific antagonistic activity, aight antibiotic resistance genes, and also three CRISPR blocks and eight Cas I-B system genes were identified in the genomes of the both strains. The expression of some genes was confirmed. In fact, the presence of identified genes suggests that L. helveticus D75 and D76 are able to form biofilms on the outer mucin layer, inhibit the growth of pathogens and pathobionts, utilize milk substrates with the formation of digestible milk sugars and bioactive peptides, resist bacteriophages and show some genome-determined resistance to antibiotics, stimulate the host’s immune system. Pathogenicity genes have not been identified. The study results confirm the safety and high probiotic potential of the strains.

Evidence of familial inheritance in non-medullary thyroid cancer (NMTC) has accumulated over the last few decades. However, known variants account for a very small percentage of the genetic burden. Here, we focused on the identification of common pathways and networks enriched in NMTC families to better understand its pathogenesis with the final aim of identifying one novel high/moderate-penetrance germline predisposition variant segregating with the disease in each studied family. We performed whole genome sequencing on 23 affected and 3 unaffected family members from five NMTC-prone families and prioritized the identified variants using our Familial Cancer Variant Prioritization Pipeline (FCVPPv2). In total, 31 coding variants and 39 variants located in upstream, downstream, 5′ or 3′ untranslated regions passed FCVPPv2 filtering. Altogether, 210 genes affected by variants that passed the first three steps of the FCVPPv2 were analyzed using Ingenuity Pathway Analysis software. These genes were enriched in tumorigenic signaling pathways mediated by receptor tyrosine kinases and G-protein coupled receptors, implicating a central role of PI3K/AKT and MAPK/ERK signaling in familial NMTC. Our approach can facilitate the identification and functional validation of causal variants in each family as well as the screening and genetic counseling of other individuals at risk of developing NMTC.

A novel binary toxin-positive non-027, non-078 Clostridium difficile strain designated LC693 whose sequence type was ST201 was isolated from the fecal sample of a patient with severe diarrhea in China. To understand the pathogenesis basis of C. difficile ST201, this recently recovered isolate LC693 was then chosen for whole genome sequencing. The project finally generated an estimated genome size of approximately 4.07 Mbp. The genome sequence was then analyzed together with the other two ST201 strains VL-0104 and VL-0391 and compared to the epidemic 027/ST1 and 078/ST11 strains. Phylogenetic analysis demonstrated that the ST201 strains belonged to clade 3. Genome size of the three ST201 strains ranged from 4.07 Mb~4.16 Mb, with an average GC content between 28.5%~28.9%. The ST201 genomes contained more than 40 antibiotic resistance genes and 15 of them were predicted to be associated with vancomycin-resistance, suggesting that they may have a strong antibiotic resistance. The ST201 strains contained a typical clade 3 specific PaLoc with a Tn6218 element inserted, and those genes harbored on their PaLoc that participated in the toxin expression and regulation were highly homologues to the epidemic 027 and 078 strains, with the exception of tcdC. A truncated TcdC was found in the ST201 strains, which is suggestive to have a contribution to the toxin production of the ST201 strains. In addition, the ST201 strains contained intact binary toxin coding and regulation genes, which is also proposed to contribute to the virulence. Genome comparison of the ST201 strains with the epidemic 027 and 078 strain identified 641 genes specific for the C. difficile ST201, and a number of them were predicted as fitness and virulence associated genes. The identification of those genes also contributes to the pathogenesis of the ST201 strain. To our knowledge, this is the first study that the genome sequence of C. difficile ST201 was discussed in detail, and the present study would have a contribution to understanding the pathogenesis basis of C. difficile ST201.

Background: After its initial detection in Wuhan, China, in December 2019, SARS-CoV-2 has spread rapidly, causing successive epidemic waves worldwide. This study aims to provide a genomic epidemiology of SARS-CoV-2 in Burkina Faso. Methods: Three hundred and seventy-seven SARS-CoV-2 genomes obtained from PCR-positive nasopharyngeal samples (PCR cycle threshold score <35) collected between May 5, 2020, and January 31, 2022 were analysed. Genomic sequences were assigned to phylogenetic clades using NextClade and to Pango lineages using pangolin. Phylogenetic and phylogeographic analyses were performed to determine the geographical sources and time of virus introduction in Burkina Faso. Results: The analyzed SARS-CoV-2 genomes could be assigned to 10 phylogenetic clades and 27 Pango lineages already described worldwide. Our analyses revealed the important role of cross-border human mobility in the successive SARS-CoV-2 introductions in Burkina Faso from neighboring countries. Conclusion: This study provides additional insights into the genomic epidemiology of SARS-CoV-2 in West Africa. It highlights the importance of land travel in the spread of the virus and the need to rapidly implement preventive policies. Regional cross-border collaborations and the adherence of the general population to government policies are key to prevent new epidemic waves.

Clinical metagenomics (CMg), referred to as the application of next-generation sequencing (NGS) to clinical samples, is a promising tool for the diagnosis of hospital-acquired pneumonia (HAP). Indeed, CMg allows identifying pathogens and antibiotic resistance genes (ARGs), thereby providing the information required for the optimization of the antibiotic regimen. Hence, provided that CMg would be faster than conventional culture, the probabilistic regimen used in HAP could be tailored faster, which should lead to an expected decrease of mortality and morbidity. While the inference of the antibiotic susceptibility testing from metagenomic or even genomic data is challenging, a limited number of antibiotics are used in the probabilistic regimen of HAP (namely beta-lactams, aminoglycosides, fluoroquinolones, glycopeptides and oxazolidinones). Accordingly in the perspective of applying CMg to the early diagnostic of HAP, we aimed at reviewing the performances of whole genomic sequencing (WGS) of the main HAP-causing bacteria (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia and Staphylococcus aureus) for the prediction of susceptibility to the antibiotic families advocated in the probabilistic regimen of HAP.

The holistic approach of One Health, which sees human, animal, plant, and environmental health as a unit, rather than discrete parts, requires not only interdisciplinary cooperation, but standardized methods for communicating and archiving data, enabling participants to easily share what they have learned and allow others to build upon their findings.Ongoing work by NCBI and the GenomeTrakr project illustrates how open data platforms can help meet the needs of federal and state regulators, public health laboratories, departments of agriculture, and universities. Here we describe how microbial pathogen surveillance can be transformed by having an open access database along with Best Practices for contributors to follow. First, we describe the open pathogen surveillance framework, hosted on the NCBI platform. We cover the current community standards for WGS quality, provide an SOP for assessing your own sequence quality and recommend QC thresholds for all submitters to follow. We then provide an overview of NCBI data submission along with step by step details. And finally, we provide curation guidance and an SOP for keeping your public data current within the database. These Best Practices can be models for other open data projects, thereby advancing the One Health goals of Findable, Accessible, Interoperable and Re-usable (FAIR) data.

A previous study involving whole genome sequencing of the white shark suggested unique molecular evolution accounting for gigantism and the enhanced longevity of sharks including positive selection of dozens of protein-coding genes potentially involved in genome stability. We performed a reanalysis on some of the genes and identified serious flaws in their results. In this short article, we scrutinize one of the serious problems we identified, report other concerns, and point out a potential bias in analyzing iconic shark species in general.

Recently the polyvalent bacteriophage FP01, isolated from wastewater in Valparaiso, Chile, was described to have lytic activity across species against Escherichia coli and Salmonella enterica serovars. Due to it polyvalent nature the bacteriophage FP01 could have potential application in food and agri-industry. Also, fundamental aspects of polyvalent bacteriophage biology are not well known. In this study we sequenced and describe the complete genome of the polyvalent phage FP01 (MH745368) using the nanopore technology. The bacteriophage FP01 genome has a 44,900 bp, double-stranded DNA with an average G+C content of 49.41% and 90 coding sequences (CDSs). We found that the phage FP01 critically depends on host factors for replication and transcription. Also, it has a critical lysogenic repressor pseudogene. Phylogenetic analyses indicated that the phage FP01 is closely related to phages lambda and P22. These results suggest that the phage FP01 could be a lytic variant of a lysogenic phage or acquired genes from lysogenic phages during host infection.

We performed a comprehensive analysis of angiosarcoma (AS) genomic biomarkers and their associations with the site of origin. We aimed to describe the genomic landscape of AS in a cohort of 143 cases of AS profiled by Caris Life Sciences. Data of Next Generation Sequencing (NGS) with a 592 gene panel was available for the entire cohort. Fifty-three cases had data of Whole Exome Sequencing (WES) which we used to study the microenvironment phenotype. Immuno-therapy (IO) response biomarkers: Tumor Mutation Burden (TMB), Microsatellite Instability (MSI) and PD-L1 status were included. IO-response markers were present in 36.4% of the cohort and in 65% of head and neck AS (H/N-AS) (p<0.0001). H/N-AS cases had predominantly muta-tions in TP53 (50.0%, p=0.0004), POT1 (40.5%, p<0.0001) and ARID1A (33.3%, p=0.5875). In breast AS, leading alterations were MYC amplification (63.3%, p<0.0001), HRAS (16.1%, p=0.0377), and PI3KCA (16.1%, p=0.2352). A microenvironment with a high immune signature, associated with better response to IO, was present in 13% of the cases. This signature was evenly distributed among different primary sites. We found that the molecular biology for AS varies significantly according to the primary site. Our findings can facilitate the design and optimiza-tion of therapeutic strategies for AS to overcome resistance to IO and targeted therapies.

Ganoderma boninense is the major causal agent for the basal stem rot (BSR) disease in oil palm, causing the progressive rot of the basal part of the stem. Despite its prominence, key pathogenicity determinants for the aggressive nature of hemibiotrophic infection remain unknown. In this study, genome sequencing and annotation of G. boninense T10 were carried out using the Illumina sequencing platform and comparative genome analysis was performed with previously reported G. boninense strains (NJ3 and G3). The pan-secretome of G. boninense was constructed and comprised of 937 core orthogroups, 243 accessory orthogroups, and 84 strain-specific orthogroups. A set of core candidate effector proteins (CEPs) were found to be enriched with catalytic protein classified as the carbohydrate-active enzymes, hydrolases as well as non-catalytic proteins. Differential expression analysis revealed an upregulation of CEP genes which was linked to the suppression of PTI signaling cascade while the downregulation of CEP genes was linked to the inhibition of PTI by preventing host defense elicitation. Genome architecture analysis revealed the one-speed architecture of the G. boninense genome and the lack of preferential association of CEP genes to the transposable elements. The findings obtained from this study would aid in the characterization of pathogenicity determinants and molecular biomarkers of BSR disease.

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently pose a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carries mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n=586). mcr-1 genes were detected using a previously developed qPCR assay and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological background.

As exemplified by the global response to the SARS-CoV-2 pandemic, whole genome sequencing played an important role in monitoring the evolution of novel viral variants and provided guidance on potential antiviral treatments. The recent rapid and extensive introduction and spread of highly pathogenic avian influenza virus in Europe, North America and elsewhere raises the need for similarly rapid sequencing to aid in appropriate response and mitigation activities. To facilitate this objective, we investigated a next generation sequencing platform that uses a portable nanopore sequencing device to generate and present data in real time. This platform offers the potential to extend in-house sequencing capacities to laboratories that may otherwise lack resources to adopt sequencing technologies requiring large benchtop instruments. We evaluated this platform for routine use in a diagnostic laboratory. In this study we evaluated different primer sets for the whole genome amplification of influenza A virus and evaluated five different library preparation approaches for sequencing on the nanopore platform using the MinION flow-cell. A limited amplification procedure and a rapid procedure were found to be best among the approaches taken.

Closely related to the model plant Arabidopsis thaliana, the genus Boechera is known to contain both sexual and apomictic species or accessions. Boechera retrofracta is a diploid sexually reproducing species and is thought to be an ancestral parent species of the apomictic species Boechera divaricarpa. Here we report the de novo assembly of the B. retrofracta genome using short Illumina and Roche reads from 1 paired-end and 3 mate pair libraries. The distribution of 23-mers from the paired end library has indicated a low level of heterozygosity and the presence of detectable duplications and triplications. The genome size was estimated to be equal 227 Mb. N50 of the assembled scaffolds was 2.3 Mb. 27048 protein-coding genes were predicted using a hybrid approach that combines homology-based and de novo methods. Also repeats, tRNA and rRNA genes were annotated. Finally, genes of B. retrofracta and 6 other Brassicaceae species were used for phylogenetic tree reconstruction. Also, a detailed analysis of evolution of the APOLLO apomixis-associated locus was performed. An assembled genome of B. retrofracta will help in the challenging assembly of the highly heterozygous genomes of hybrid apomictic species such as B. divaricarpa.

Newcastle disease virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to investigate the genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and swans were collected from different geographic regions within 13 governorates during 2019-2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing hemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV spread to 12 governorates in domestic birds, migratory and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70 - 99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, based on the presence of polybasic amino acids (RRQRF) at the F gene cleavage site. Phylogenetic analysis revealed that the NDV strains belonged to class II within genotype VII.1.1. The presence of genetically similar virulent NDV in wild birds further highlights their role in the dissemination of NDV in poultry populations across Egypt. Continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV incursions and genetic diversification.

Recent evidence indicates that the risk of being diagnosed with cancer in a tissue is strongly correlated (0.80) with the number of stem cell divisions accumulated by the tissue. Since cell division can generate random mutations during DNA replication, this correlation has been used to propose that cancer is largely caused by the accumulation of unavoidable mutations in driver genes. However, no correlation between the number of gene mutations and cancer risk across tissues has been reported. Because many somatic mutations in cancers originate prior to tumor initiation and the number of cell divisions occurring during tumor growth is similar among tissues, here I use whole genome sequencing information from 22,086 cancer samples and incidence data from the largest cancer registry in each continent to study the relationship between the number of gene mutations and the risk of cancer across 33 tissue types. Results show a weak positive correlation (mean = 0.14) between these two parameters in each of the five cancer registries. The correlation became stronger (mean = 0.50) when gender-related cancers were excluded. Results also show that 1,003 samples from 29 cancer types have zero mutations in genes. These data suggest that cancer etiology can be better explained by the accumulation of stem cell divisions than by the accumulation of gene mutations. Possible mechanisms by which the accumulation of cell divisions in stem cells increases the risk of cancer are discussed.

Young women with breast cancer represent 15% of cancer cases in Latin America. Genomic studies have found that early-onset breast-cancer cases exhibit a higher genetic susceptibility and a specific genomic signature as compared to their older counterparts. The aim of this study was to describe clinically relevant germline mutations in a cohort of young women with breast cancer. To achieve this, we analyzed hereditary-cancer genes from whole-exome sequencing data in 108 unrelated women with an extreme phenotype of breast cancer (≤40 years of age), diagnosed and treated at the National Cancer Institute of Mexico; 11% of the patients carried a pathogenic variant. BRCA2 comprised 46% of the mutations, followed by BRCA1 with 23%; PALB2 with 15%; and TP53 and RAD51C with 8 % each. This article describes a novel pathogenic mutation in RAD51C c.519dupT. The median age at diagnosis was 35 years overall; however, it was six years younger in patients with mutations. Age at diagnosis (OR=0.82, CI 95% 0.71-0.94; P= 0.008) and first-degree family history of cancer (OR=8.26, CI95% 1.35-50; P= 0.022) were the only epidemiological variables associated with mutational status. We found no differences in disease-free survival (p=0.403) or overall survival (p=0.735) among mutational status subgroups.

Despite advances in algorithms and computational platforms, de-novo genome assembly remains a challenging process. Due to the constant innovation in sequencing technologies (Sanger, SOLiD, Illumina, 454, PacBio and Oxford Nanopore), genome assembly has evolved to respond to the changes in input data type. This paper includes a broad and comparative review of the most recent short-read, long-read and hybrid assembly techniques. In this review, we provide (1) an algorithmic description of the important processes in the workflow that introduces fundamental concepts and improvements; (2) a review of existing software that explains possible options for genome assembly; and (3) a comparison of the accuracy and the performance of existing methods executed on the same computer using the same processing capabilities and using the same set of real and synthetic datasets. Such evaluation allows a fair and precise comparison of accuracy in all aspects. As a result, this paper identifies both the strengths and weaknesses of each method. This comparative review is unique in providing a detailed comparison of a broad spectrum of cutting-edge algorithms and methods.

Development and deployment of wheat varieties with high yields, wide adaptability, good quality, multiple-resistance to abiotic and biotic stresses, and efficient response to fertilizers have greatly contributed to global wheat sustainable production. The genomic composition of key commercial wheat variety can help understand the genetic basis underlying the development of new variety and permit increased breeding efficiency. In this study, we report the chromosomal and genomic compositions of BN207, presently the leading wheat variety in the southern region of Huang-Huai River Valley, the most important wheat producing area in China through an integrated analysis using fluorescent in situ hybridization (FISH) and wheat 15 K SNP array. Our results showed that BN207 inherited 55.3% and 40.7% of its genome from its male parent BN64 and female parent ZM16, respectively, and generating 64 novel or recombined loci. Besides, we detected nine chromosomal variations in Bn207 and its parents and ten sister lines, and physically mapped two variations, the pericentric inversion of chromosome 6B, and large tandem repeat sequence block at the long arm of 5A, both had positive effects on agronomic traits, by integration of FISH and SNP loci recombination analyses. These results will provide a reference for breeding of high yield wheat varieties as BN207, and the application of founder parents BN64 and ZM16, which are being utilized frequently in wheat breeding programs in Henan Province and surrounding areas.

The SnRK gene family is a key regulator playing an important role in plant stress response by phosphorylating the target protein to regulate the signalling pathways. The function of SnRK gene family has been reported in many species but is limited to Triticum asetivum. In this study, SnRK gene family in the wheat genome was identified and its structural characteristics were described. One hundred forty-seven SnRK genes distributed across 21 chromosomes were identified in the Triticum aestivum genome and categorised into three subgroups (SnRK1/2/3) based on phylogenetic analyses and domain types. The gene intron-exon structure and protein-motif composition of SnRKs were similar within each subgroup but different amongst the groups. Gene duplication between the wheat, Arabidopsis, rice and barley genomes was also investigated in order to get insight into the evolutionary aspects of the TaSnRK family genes. SnRK genes showed differential expression patterns in leaves, roots, spike, and grains. Redundant stress-related cis-elements were also found in the promoters of 129 SnRK genes and their expression levels varied widely following drought, ABA and light regulated elements. In particular, TaSnRK2.11 had higher and increased expression under the abiotic stresses and can be a candidate gene for the abiotc stress tolerance. The findings will aid in the functional characterization of TaSnRK genes for further research.

Sweet cucumber (Solanum muricatum) sect. Basarthrum, is a neglected horticultural crop native of the Andean region. It is naturally distributed very close to potatoes (Solanum sect. Petota) and tomatoes (Solanum sect. Lycopersicon), two groups of high economic importance. To date, molecular tools for this crop are still undetermined. We here obtained the first complete chloroplast (cp) genome of sweet cucumber and compared with seven Solanaceae species. Pair-end clean reads were obtained by PE 150 library and the Illumina HiSeq 2500 platform. The complete cp genome of S. muricatum had a 155,681 bp with typical quadripartite structure, containing a large single copy (LSC) region (86,182 bp) and a small single-copy (SSC) region (18,360 bp), separated by two inverted repeat (IR) regions (25,568 bp). The annotation of chloroplast genome predicted 88 protein-coding genes (CDS), 8 ribosomal RNA (rRNA) genes, 37 transfer RNA (tRNA) genes, and one pseudogene. A total of 48 perfect microsatellites were identified, divided in mononucleotide repeats (32), followed by tetranucleotide (6) and dinucleotides (5). SSRs with trinucleotides repeats (3), pentanucleotide (1) and hexanucleotide (1) repeats motifs in these genomes were identified in lower quantity. Most of these repeats were distributed in the noncoding regions. Whole chloroplast genome comparison with the other seven Solanaceae species revealed that the small and large single copy regions showed more divergence than inverted regions. Finally, phylogenetic analysis resolved that S. muricatum is a sister species to members of sections Petota + Lycopersicum + Etuberosum. This study reports for the first time the genome organization, gene content, and structural features of the cp genome of S. muricatum. Also, this study may provide the basis for evaluating genetic diversity within Solanum, and will be useful to examine the evolutionary processes in sweet cucumber landraces.

Abstract. The increasing frequency of isolation of methicillin-resistant Staphylococcus aureus (MRSA) limits the chances of effective antibacterial therapy of staphylococcal diseases and results in development of persistent infection such as bacteremia and osteomyelitis. The aim of this study was to identify features of the MRSAST239 0943-1505-2016 (SA943) genome, that contribute to the formation of both acute and chronic musculoskeletal infections. The analysis was performed using comparative genomics data of the dominant epidemic S. aureus lineages namely ST1, ST8, ST30, ST36, ST239. SA943 genome encodes proteins that provide resistance to the host immune system, suppress immunological memory and form biofilms. The molecular mechanisms of adaptation responsible for development of persistent infection were as follows: amino acid substitution in PBP2 and PBP2a, providing resistance to ceftaroline; loss of a large part of prophage DNA and restoration of nucleotide sequence of beta-hemolysin, that greatly facilitates escape of phagocytosed bacteria from phagosome and formation of biofilms; dysfunction of the AgrA system due to the presence of psm-mec and several amino acid substitutions in the AgrC; partial deletion of nucleotide sequence in genomic island vSAβ resulting in the loss of two proteases of Spl - operon; deletion of SD repeats in SdrE amino acid sequence.

To assess the genetic diversity of circulating dengue virus 2 in Senegal in 2018 we performed molecular characterization by complete genome sequencing and performing phylogenetic analysis. Sequenced strains belong to Cosmopolitan genotype of DENV-2 we observed intra-genotype variability leading to a divergence in two clades with differential geographic distribution. We report two variants namely; the “Northern variant” harbouring three nonsynonymous mutations (V1183M, R1405K, P2266T) located respectively on NS2A, NS2B and NS4A and the “Western variant” with two nonsynonymous mutations (V1185E, V3214E) located respectively in the NS2A gene and the NS5 gene. Findings calls for in depth in vitro and functional study to elucidate the impact of observed mutations on viral fitness, spread, epidemiology and disease outcome.

The domestication and subsequent genetic improvement of wheat led to the development of large-seeded cultivated wheat species relative to their smaller-seeded wild progenitors. While increased grain weight (GW) continues to be an important goal of many wheat breeding programs, few genes underlying this trait have been identified despite an abundance of studies reporting quantitative trait loci (QTLs) for GW. Here we perform a QTL analysis for GW using a population of recombinant inbred lines (RILs) derived from the cross between wild emmer wheat accession ‘Zavitan’ and durum wheat variety ‘Svevo’. Identified QTLs in this population were anchored to the recent Zavitan reference genome, along with previously published QTLs for GW in tetraploid wheat. This genome-based, meta-QTL analysis enabled the identification of a locus on chromosome 6A whose introgression from wild wheat positively affects GW. The locus was validated using an introgression line carrying the 6A GW QTL region from Zavitan in a Svevo background, resulting in >8% increase in GW compared to Svevo. Using the reference sequence for the 6A QTL region, we identified a wheat ortholog to OsGRF4, a rice gene previously associated with GW. The coding sequence of this gene (TtGRF4-A) contains four SNPs between Zavitan and Svevo, one of which reveals the Zavitan allele to be rare in a core collection of wild emmer and completely absent from the domesticated emmer genepool. Similarly, another wild emmer accession (G18-16) was found to carry a rare allele of TtGRF4-A that also positively affects GW and is characterized by a unique SNP absent from the entire core collection. These results exemplify the rich genetic diversity of wild wheat, posit TtGRF4-A as a candidate gene underlying the 6A GW QTL, and suggest that the natural Zavitan and G18-16 alleles of TtGRF4-A have potential to increase wheat yields in breeding programs.

Pantoea stewartii subsp. indologenes (Psi) is a causative agent of leafspot of foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onion. Our recent host range evaluation study identified two pathovars; P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. that are pathogenic on onion and millets or on millets only, respectively. In the current study we developed a pan-genome using the whole genome sequencing of newly identified/classified Psi strains from both pathovars [pv. cepacicola (n= 4) and pv. setariae (n=13)]. The full spectrum of the pan-genome contained 7,030 genes. Among these, 3,546 (present in genomes of all 17 strains) were the core genes that were a subset of 3,682 soft-core genes (present in ≥16 strains). The accessory genome included 1,308 shell genes and 2,040 cloud genes (present in ≤ 2 strains). The pan-genome showed a clear liner progression with >6,000 genes, suggesting the pan-genome of Psi is open. Comparative phylogenetic analysis showed differences in phylogenetic clustering of Pantoea spp. using PAVs/wgMLST approach in comparison to core genome SNP-based phylogeny. Further, we conducted a horizontal gene transfer (HGT) study including four other Pantoea species namely, P. stewartii subsp. stewartii LMG 2715T, P. ananatis LMG 2665T, P. agglomerans LMG L15, and P. allii LMG 24248T. A total of 317 HGT events among four Pantoea species were identified with most gene transfers observed between Psi pv. cepacicola and Psi pv. setariae. Pan-GWAS analysis predicted a total of 154 genes including seven cluster of genes associated with the pathogenicity phenotype on onion. One of the clusters contain 11 genes with known functions and are found to be chromosomally located.

Paper-based sensors, microfluidic platforms and electronic devices have attracted attention in the past couple of decades because they are flexible, can be recycled easily, environmentally friendly, and inexpensive. Here we report a paper aptamer-based potentiometric sensor to detect the whole Zika virus for the first time with a minimum sensitivity of 2.6 nV/Zika and the minimum detectable signal (MDS) of 0.8x1e6 Zika. Our paper sensor works very similar to a P-N junction where a junction is formed between two different wet regions with different electrochemical potentials near each other on the paper. These two regions with slightly different ionic contents, ionic species and concentrations, produce a potential difference given by the Nernst equation. Our paper sensor consisted of a 2-3 mm x 10 mm segments of a paper with a conducting silver paint contact patches on its two ends. The paper is soaked in a buffer solution containing aptamers designed to bind to the capsid proteins on Zika. Atomic force microscopy studies were carried out to show both the aptamer and Zika become immobilized in the paper. We then added the Zika (in its own buffer or simulant Urine) to the region close to one of the silver-paint contacts. The Zika virus (40 nm diameter with 43 kDa or 7.1x10-20 gm weight), became immobilized in the paper’s pores and bonded with the resident aptamers creating a concentration gradient. The potential measured between the two silver paint contacts reproducibly became more negative as upon adding the Zika. We also showed that an LCD powered by the sensor, can be used to detect the sensor output.

Paper-based sensors, microfluidic platforms and electronic devices have attracted attention in the past couple of decades because they are flexible, can be recycled easily, environmentally friendly, and inexpensive. Here we report a paper aptamer-based potentiometric sensor to detect the whole Zika virus for the first time with a minimum sensitivity of 2.6 nV/Zika and the minimum detectable signal (MDS) of 1.2x10⁶ Zika. Our paper sensor works very similar to a P-N junction where a junction is formed between two different wet regions with different electrochemical potentials near each other on the paper. These two regions with slightly different ionic contents, ionic species and concentrations, produce a potential difference given by the Nernst equation. Our paper sensor consisted of a 2-3 mm x 10 mm segments of a paper with a conducting silver paint contact patches on its two ends. The paper is soaked in a buffer solution containing aptamers designed to bind to the capsid proteins on Zika. Atomic force microscopy studies were carried out to show both the aptamer and Zika become immobilized in the paper. We then added the Zika (in its own buffer) to the region close to one of the silver-paint contacts. The Zika virus (40 nm diameter with 43 kDa or 7.1x10^-20 gm weight), became immobilized in the paper’s pores and bonded with the resident aptamers creating a concentration gradient. The potential measured between the two silver paint contacts reproducibly became more negative as upon adding the Zika. We also showed that an LCD powered by the sensor, can be used to detect the sensor output.

The use of circulating cell-free (cf) DNA to monitor cancer progression and response to therapy has significant potential but there is only limited data on whether this technique can detect the presence of low frequency subclones that may ultimately confer therapy resistance. In this study, we sought to evaluate whether whole-exome sequencing of cfDNA can accurately profile the mutation landscape of metastatic melanoma. We used whole-exome sequencing (WES) to identify variants in matched tumor-derived genomic (g) DNA and plasma-derived cfDNA isolated from a cohort of 10 metastatic cutaneous melanoma patients. WES parameters such as sequencing coverage and total sequencing reads were comparable between gDNA and cfDNA. There was significant concordance between gDNA and cfDNA based on the total number of variants identified and the degree of overlap in variants which was independent of the site of tumor biopsy. The mutant allele frequency of common single nucleotide variants was lower in cfDNA reflecting lower read depth and dilution of circulating tumor DNA in the circulation by other cfDNA species. In addition to known melanoma driver mutations, several other melanoma-associated mutations were found to be concordant between matched gDNA and cfDNA. This study highlights that WES of cfDNA can capture clinically-relevant mutations present in melanoma metastases, but does not appear to provide any additional unique information on tumor heterogeneity. Targeted deep sequencing may be required to detect low frequency genomic aberrations known for predicting therapy resistance.

Next-generation sequencing is now a mature technology, allowing partial animal genomes to be produced for many clades. Though many software exist for genome assembly and annotation, a simple pipeline that allows researchers to input raw sequencing reads in fastq format and allow the retrieval of a completely assembled and annotated mitochondrial genome is still missing. mitoMaker 1.0 is a pipeline developed in python that implements (i) recursive de novo assembly of mitochondrial genomes using a set of increasing k-mers; (ii) search for the best matching result to a target mitogenome and; (iii) performs iterative reference-based strategies to optimize the assembly. After (iv) checking for circularization and (v) positioning tRNA-Phe at the beginning, (vi) geneChecker.py module performs a complete annotation of the mitochondrial genome and provides a GenBank formatted file as output.

The chloroplast genome is conservative and stable, which can be employed to resolve genotypes. Currently, published nuclear sequences and molecular markers failed to differentiate the species from taxa robustly, including Machilus leptophylla, Hanceola exserta, Rubus bambusarum, and Rubus henryi. In this study, the four chloroplast genomes were characterized, and then their simple sequence repeats (SSRs) and phylogenetic positions were analyzed. The results demonstrated the four chloroplast genomes consisted of 152.624 kb, 153.296kb, 156.309 kb, and 158.953 kb in length, involving 124, 130, 129, and 131 genes, respectively. Moreover, the chloroplast genomes contained typical four regions. Six classes of SSR were identified from the four chloroplast genomes, in which mononucleotide was the class with the most members. The types of the repeats were various within individual classes of SSR. Phylogenetic trees indicated that M. leptophylla was clustered with M. yunnanensis, and H. exserta was confirmed under family Ocimeae. Additionally, R. bambusarum and R. henryi were clustered together, whereas they did not belong to the same species due to the differing SSR features. This research would provide evidence for resolving the species and contributed new genetic information for further study.

In addition to the risk of vaccine failure caused by strain mismatch, the production of inactivated FMD vaccines is dangerous if adequate biosafety cannot be maintained. Using a high-throughput sequencing protocol optimized for short nucleic acid fragments, the composition of a local inactivated vaccine was analyzed in depth. The serotype O strain identified in the vaccine was genetically identical to viruses found in recent FMD outbreaks in Egypt.

The genus Fritillaria belongs to the widely distributed family Liliaceae. The bulbs of Fritillaria ussuriensis and Fritillaria cirrhosa are valuable herbaceous medicinal ingredients. However, they are still used indiscriminately in herbal medicine. Identification and molecular phylogenic analysis of Fritillaria species is therefore required. Here, we report the complete chloroplast (cp) genome sequences of F. ussuriensis and F. cirrhosa. The two Fritillaria cp genomes were 151,524 and 151,083 bp in length, respectively, including a pair of inverted repeat regions (52,678 and 52,156 bp) separated by a large single copy region (81,732 and 81,390 bp) and small single copy region (17,114 and 17,537 bp). A total of 111 genes in F. ussuriensis and 112 in F. cirrhosa comprised 77 protein-coding genes in F. ussuriensis and 78 in F. cirrhosa, 30 tRNA genes, and four rRNA genes. The gene order, content, and orientation of the two Fritillaria cp genomes exhibited the general structure of flowering plants, and were similar to those of other Fritillaria species. Comparison of the six Fritillaria species’ cp genomes indicated seven highly divergent regions in intergenic spacers and in the matK, rpoC1, rpoC2, ycf1, ycf2, ndhD, and ndhF coding regions. We established the position of the six species through phylogenic analysis. The complete chloroplast genome sequences of two Fritillaria species will be useful genomics resources for identification of Fritillaria species and for studying the phylogenetic relationship among Fritillaria species within the Liliaceae family.

CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly es-tablished over the past decade. Ironically, the emergence of numerous methods makes the right choice of method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans and illustrates key strategies. Our overview of significant advances in CRISPR-Cas will help readers to understand current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.

Fusarium circinatum is an important global pathogen of pine trees. Genome plasticity has been observed in different isolates of the fungus, but no genome comparisons are available. To address this gap, we sequenced and assembled to chromosome level five isolates of F. circinatum. These genomes were analysed together with previously published genomes of F. circinatum isolates FSP34 and KS17. Multi-sample variant calling identified a total of 461683 micro variants (SNPs and small indels) and a total of 1828 macro structural variants of which 1717 were copy number variants and 111 were inversions. Variant density was higher on sub-telomeric regions of chromosomes. Variant annotation revealed that genes involved in transcription, transport, metabolism and transmembrane proteins were overrepresented in gene sets affected by high impact variants. A core genome representing genomic elements conserved in all the isolates and a non-redundant pangenome representing all genomic elements is presented. Whole genome alignments showed that an average of 93% of the genomic elements are present in all isolates. The results of this study reveal that some genomic elements are not conserved within the isolates and some variants are high impact. The described genome-scale variations will help inform novel disease management strategies against the pathogen.

Family Phyllanthaceae is one of the largest segregates of the eudicot order Malpighiales and its species are herb, shrub, and tree, which are mostly distributed in tropical regions. Certain taxonomic discrepancies exist at genus and family level. Here, we report chloroplast genomes of three Phyllanthaceae species—Phyllanthus emblica, Flueggea virosa, and Leptopus cordifolius— and compare them with six others previously reported Phyllanthaceae chloroplast genomes. The species of Phyllanthaceae displayed quadripartite structure, comprising inverted repeat regions (IRa and IRb) that separate large single copy (LSC) and small single copy (SSC) regions. The length of complete chloroplast genome ranged from 154,707 bp to 161,093 bp; LSC from 83,627 bp to 89,932 bp; IRs from 23,921 bp to 27,128 bp; and SSC from 17,424 bp to 19,441 bp. Chloroplast genomes contained 111 to 112 unique genes, including 77 to 78 protein-coding, 30 transfer RNA (tRNA), and 4 ribosomal RNA (rRNA) that showed similarities in arrangement. The number of protein-coding genes varied due to deletion/pseudogenization of rps16 genes in Baccaurea ramiflora and Leptopus cordifolius. High variability was seen in number of oligonucleotide repeats while analysis of guanine-cytosine (GC) content, codon usage, amino acid frequency, simple sequence repeats analysis, synonymous and non-synonymous substitutions, and transition and transversion substitutions showed similarities in all Phyllanthaceae species. We detected a higher number of transition substitutions in the coding sequences than non-coding sequences. Moreover, the high number of transition substitutions was determined among the distantly related species in comparison to closely related species. Phylogenetic analysis shows the polyphyletic nature of the genus Phyllanthus which requires further verification. We also determined suitable polymorphic coding genes, including rpl22, ycf1, matK, ndhF, and rps15 which may be helpful for the reconstruction of the high-resolution phylogenetic tree of the family Phyllanthaceae using a large number of species in the future. Overall, the current study provides insight into chloroplast genome evolution in Phyllanthaceae.

Nyssa yunnanensis is a deciduous tree species in the family Nyssaceae within the order Cornales. As only eight individual trees and two populations have been recorded in China’s Yunnan province, this species has been listed among China’s national Class I protection species since 1999 and also among 120 PSESP (Plant Species with Extremely Small Populations) in the Implementation Plan of Rescuing and Conserving China’s Plant Species with Extremely Small Populations(PSESP) (2011-2-15). Here, we present the draft genome assembly of N. yunnanensis. Using 10X Genomics linked-reads sequencing data, we carried out the de novo assembly and annotation analysis. The N. yunnanensis genome assembly is 1475 Mb in length, containing 288,519 scaffolds with a scaffold N50 length of 985.59 kb. Within the assembled genome, 799.51 Mb was identified as repetitive elements, accounting for 54.24% of the sequenced genome, and a total of 39,803 protein-coding genes were predicted. With the genomic characteristics of N. yunnanensis available, our study might facilitate future conservation biology studies to help protect this extremely threatened tree species.

The recent report that DNA extracted from ancient bone must have from the offspring of a female Neanderthal and a male Denisovan depends on the inference that the subject has a high level of heterozygosity for Neanderthal and Denisovan alleles across the genome. Here I point out that the relative frequencies of derived transversion polymorphisms varies markedly between the new specimen, Denisova 11, and two high-coverage Neanderthal genomes. In Denisova 11 the AC and CG polymorphisms are much commoner than the others and are almost twice as common as the AT polymorphism. In the high-coverage Neanderthal genomes the four types of transversion are about equally common, with the AT being slightly commoner than the others. These results suggest that allele-calling errors are frequent and that this may provide an alternative explanation for the observed heterozygosity.

Plasma and serum are the most widely used blood-derived biofluids for metabolomics and lipidomics assays, but the isolation of these products from blood may introduce additional bias as indicated by the fact that many analytes that are present at high concentrations in blood cells cannot be measured and evaluated in those samples. Of particular concern, variable hemolysis during the pre-processing of blood products could compromise accurate and reproducible quantification. Compared with plasma or serum, whole blood may be a better alternative due to simplicity of processing. In this study, we provide a comprehensive method for quantification of the whole blood sphingolipidome and the concentrations were compared with those from plasma. Combining a single-phase extraction method with liquid-chromatography high resolution mass spectrometry (R=120, 000), assisted by alkaline hydrolysis, we were able to identify and simultaneously quantify more than 150 sphingolipids. Furthermore, most of sphingolipids remained stable after a freeze/thaw cycle. Whole blood contained a higher concentration of most sphingolipids than corresponding plasma. Moreover, individual variations in the levels of sphingolipids were lower for whole blood than plasma. These findings demonstrate that whole blood could be a better alternative to plasma, and potentially guide the evaluation of sphinglipidome for biomarker discovery.

About 15% of colorectal cancer (CRC) patients have first-degree relatives affected by the same malignancy. However, for most families the cause of familial aggregation of CRC is unknown. In order to identify novel high-to-moderate penetrant germline variants underlying CRC susceptibility, we performed whole exome sequencing (WES) on four CRC cases and two unaffected family members of a Polish family without any mutation in known CRC predisposition genes. After WES, we used our in-house developed Familial Cancer Variant Prioritization Pipeline and identified two novel variants in the solute carrier family 15 member 4 (SLC15A4) gene. The heterozygous missense variant, p. Y444C, was predicted to affect the phylogenetically conserved PTR2/POT domain and to have a deleterious effect on the function of the encoded peptide/histidine transporter. The other variant was located in the upstream region of the same gene (GRCh37.p13, 12_129308531_C_T; 43bp upstream of transcription start site, ENST00000266771.5) and it was annotated to affect the promoter region of SLC15A4 as well as binding sites of 17 different transcription factors. Our findings of two distinct variants in the same gene may indicate a synergistic up-regulation of SLC15A4 as the underlying genetic cause and implicate this gene for the first time in genetic inheritance of familial CRC.

Purpose: Novel coronavirus disease (COVID-19) is the current global concern. Radiotherapy (RT), commonly employed in cancer management, has been considered one of the potential treatments for COVID-19 pneumonia. Here, we present the final report of the pilot trial evaluating the efficacy and safety of low-dose whole-lung irradiation (LD-WLI) in patients with COVID-19 pneumonia. Methods and Materials: We enrolled patients with moderate COVID-19 pneumonia who were older than 60 years. Participants were treated with LD-WLI in a single fraction of 0.5 or 1.0Gy along with the national protocol of COVID-19. The primary endpoints were improvement of SpO₂, the number of hospital/ICU stay days, and the number of intubations after RT and the secondary endpoints were alterations of the c-reactive peptide, interleukin-6, ferritin, procalcitonin, and D-dimer. The response rate (RR) was defined as a rise in SpO₂ upon RT with rising or constant trend in the next two days, and clinical recovery (CR) included patients who were discharged from the hospital or acquired SpO₂ ≥93% on room air. Results: Between 21 May 2020 and 2 July 2020, ten patients were enrolled. The median age was 75 years, 80% were male, and 80% had comorbidities. The first five patients received a single 0.5Gy-WLI, and others received 1.0Gy. Patients were followed for 2-14 days (median 5.5 days). Following one day, nine patients experienced an improvement in SpO₂. Five patients were discharged (median 6^th day, range 2^nd-14^th day), and four patients died (median 7^th day, range 3^rd-10^th day). Overall, the RR and CR were 60.0% and 55.5%, respectively. The RR and CR rates of 0.5- and 1.0Gy group were 80% vs 40% and 75% vs 40%, respectively. No acute radiation-induced toxicity was recorded. Conclusions: LD-WLI with a single 0.5Gy fraction seems to be a more appropriate dose to warrant further evaluation in a large-scale, randomized trial.

Genome-wide association studies (GWAS) have identified and reproduced thousands of diseases associated loci but many of them are not directly interpretable due to the strong linkage disequilibrium among variants. Transcriptome-wide association studies (TWAS) incorporated expression quantitative trait loci (eQTL) cohorts as reference panel to detect associations with the phenotype at the gene level and were gaining popularity in recent years. For nicotine addiction, several important susceptible genetic variants were identified by GWAS, but TWAS that detected genes associated with nicotine addiction and unveiled the underlying molecular mechanism were still lacking. In this study, we used eQTL data from the Genotype-Tissue Expression (GTEx) consortium as reference panel to conduct tissue specific TWAS on cigarettes per day (CPD) over 13 brain tissues in two large cohorts: UK Biobank (UKBB; N=142,202) and the GWAS &amp; Sequencing Consortium of Alcohol and Nicotine use (GSCAN; N=143,210), and then meta-analyzed the results across tissues while considering the heterogeneity across tissues. We identified three major clusters of genes with different meta-patterns across tissues consistent in both cohorts, including homogenous genes associated with CPD in all brain tissues, partially homogeneous genes associated with CPD in cortex, cerebellum and hippocampus tissues, and lastly the tissue-specific genes associated with CPD in only few specific brain tissues. Downstream enrichment analyses on each gene cluster identified unique biological pathways associated with CPD and provided important biological insights into the regulatory mechanism of nicotine dependence in the brain.

Background: Immunoglobulin A nephropathy (IgAN) is a complex autoimmune disease, and the exact pathogenesis remains to be elucidated. Methods: We conducted summary data-based Mendelian randomization (SMR) analysis and performed functional mapping and annotation using FUMA to explore genetic loci that are po-tentially involved in the pathogenies of IgAN. Both analyses used summarized data of a recent genome-wide association study (GWAS) on IgANs, which included 477,784 Europeans (15,587 cases and 462,197 controls) and 175,359 East Asians (71 cases and 175,288 controls). We performed separate SMR analysis using CAGE and GTEx eQTL data. Results: Using the CAGE eQTL data, our SMR analysis identified 32 probes tagging 25 unique genes that were pleiotropically/potentially causally associated with IgAN, with the top three probes being ILMN_2150787 (tagging HLA-C, PSMR=2.10×10-18), ILMN_1682717 (tagging IER3, PSMR=1.07×10-16) and ILMN_1661439 (tagging FLOT1, PSMR=1.16×10-14). Using GTEx eQTL data, our SMR analysis identified 24 probes tagging 24 unique genes, with the top three probes being ENSG00000271581.1 (tagging XXbac-BPG248L24.12, PSMR=1.44×10-10), ENSG00000186470.9 (tagging BTN3A2, PSMR=2.28×10-10), and ENSG00000224389.4 (tagging C4B, PSMR=1.23×10-9). FUMA analysis identified 3 independent, significant and lead SNPs, 2 genomic risk loci and 39 genes. Conclusion: We identified many genetic variants/loci that are potentially involved in the patho-genesis of IgAN.

The IgM and IgT classes were previously identified and characterized in the Antarctic teleost Trematomus bernacchii, a species belonging to the Perciform suborder Notothenoidei. Herein we characterized the gene encoding the polymeric immunoglobulin receptor (pIgR) in the same species and compared it to pIgR of multiple teleost species belonging to five perciform sub-orders, including 11 Antarctic and one non-Antarctic (Cottoperca gobio) notothenioid species, the latter living in less cold periantarctic sea. Antarctic pIgR genes displayed particularly long in-trons marked by sites of transposable elements and transcription factors. Furthermore, analysis of T. bernacchii pIgR cDNA unveiled multiple amino acid substitutions unique to Antarctic species, all introducing adaptive features, including N-glycosylation sequons. Interestingly, C. gobio shared most features with the other perciforms rather than with the cold adapted relatives. T. bernacchii pIgR transcripts were predominantly expressed in mucosal tissues, as indicated by q-PCR and in situ hybridization analysis. These results suggest that in cold adapted species pIgR preserved its fundamental role in mucosal immune defense, although remarkable gene structure modifications occurred.

The Peruvian creole cattle (PCC) is a neglected breed, and is an essential livestock resource in the Andean region of Peru. To develop a modern breeding program and conservation strategies for the PCC, a better understanding of the genetics of this breed is needed. We sequenced the whole genome of the PCC using a paired-end 150 strategy on the Illumina HiSeq 2500 platform, obtaining 320 GB of sequencing data. The obtained genome size of the PCC was 2.77 Gb with a contig N50 of 108Mb and 92.59% complete BUSCOs. Also, we identified 40.22% of repetitive DNA of the genome assembly, of which retroelements occupy 32.39% of the total genome. A total of 19,803 protein-coding genes were annotated in the PCC genome. We downloaded proteomes and genomes of the Bovinae subfamily, and conducted a comparative analysis with our draft genome. Phylogenomic analysis showed that PCC is related to Bos indicus. Also, we identified 7,746 family genes shared among the Bovinae subfamily. This first PCC genome is expected to contribute to a better understanding of its genetics to adapt to the tough conditions of the Andean ecosystem, and evolution.

Escherichia coli phage Eco_BIFF was isolated from several laboratory stocks of E. coli K-12 MG1655 derivatives. The source of the contamination is unknown. Eco_BIFF is a lytic phage that shows effective growth inhibition of E. coli K-12. Here, we announce the complete genome sequence of Eco_BIFF, and major findings from its genome annotation.

Dynamical system tools offer a complementary approach to detailed biophysical seizure modeling, with a high potential for clinical applications. This review describes the theoretical framework that provides a basis for theorizing certain properties of seizures and for their classification according to their dynamical properties at onset and offset. We describe various modeling approaches spanning different scales, from single neurons to large-scale networks. This narrative review provides an accessible overview of this field, including non-exhaustive examples of key recent works.

This study analyses the case study of a deconstruction project called the ‘Whole House Reuse’ (WHR) which aimed, firstly, to harvest materials from a residential house, secondly, to produce new products using the recovered materials, and thirdly, to organize exhibition for the local public to promote awareness on resource conservation and sustainable deconstruction practices. The study applies characterization of recovered materials through deconstruction. In addition to the material recovery, the study assesses the embodied energy saving and greenhouse gas emission abatement of the deconstruction project. Around twelve tonnes of various construction materials were harvested through a systematic deconstruction approach, most which would otherwise be disposed to landfill in the traditional demolition approach. The study estimates that the recovered materials could potentially save around 502,158MJ of embodied energy and prevent carbon emission of around 27,029kg (CO2e). Deconstruction could eventually contribute to New Zealand’s national emission reduction targets. In addition, the project successfully engages local communities and designers to produce 400 new products using the recovered materials and exhibited to the local people. The study concludes that there is a huge prospect in regard to resource recovery, emission reduction, employment and small business opportunities using deconstruction of the old house. The socio-cultural importance of the WHR project is definitely immense; however, the greater benefits of such projects are often ignored and remain unreported to wider audiences as most of the external and environmental costs have not been considered in the traditional linear economy. It is acknowledged that under a favourable market condition and with appropriate support from local communities and authorities, deconstruction could contribute significantly to resource conservation and environmental protection despite its requirement of labour intensive efforts.

The efficacy of predicting geochemical parameters with a 2-chain workflow using spectral data as the initial input is evaluated. Spectral measurements spanning the approximate 400-25000nm spectral range are used to train a workflow consisting of a non-negative matrix function (NMF) step, for data reduction, and a random forest regression (RFR) to predict 8 geochemical parameters. Approximately 175000 spectra with their corresponding chemical analysis were available for training, testing and validation purposes. The samples and their spectral and chemical parameters represent 9399 drillcore. Of those, approximately 20000 spectra and their accompanying analysis were used for training and 5000 for model validation. The remaining pairwise data (150000 samples) were used for testing of the method. The data are distributed over 2 large spatial extents (980 km2 and 3025 km2 respectively) and allowed the proposed method to be tested against samples that are spatially distant from the initial training points. Global R2 scores and wt.% RMSE on the 150000 validation samples are Fe(0.95/3.01), SiO2(0.96/3.77), Al2O3(0.92/1.27), TiO(0.68/0.13), CaO(0.89/0.41), MgO(0.87/0.35), K2O(0.65/0.21) and LOI(0.90/1.14), given as Parameter(R2/RMSE), and demonstrate that the proposed method is capable of predicting the 8 parameters and is stable enough, in the environment tested, to extend beyond the training sets initial spatial location.

Non-vertebrate species represent about ~95% of known metazoan (animal) diversity. They remain to this day relatively unexplored genetically, but understanding their genome structure and function is pivotal for expanding our current knowledge of evolution, ecology and biodiversity. Following the continuous improvements and decreasing costs of sequencing technologies, many genome assembly tools have been released, leading to a significant amount of genome projects being completed in recent years. In this review, we examine the current state of genome projects of non-vertebrate animal species. We present an overview of available sequencing technologies, assembly approaches, as well as pre and post-processing steps, genome assembly evaluation methods, and their application to non-vertebrate animal genomes.

The newly established virus family Phenuiviridae in Bunyavirales harbors viruses infecting three kingdoms of host organisms (animals, plants, and fungi), which is rare in known virus families. Many phenuiviruses are arboviruses and replicate in two distinct hosts (e.g., insects and humans or rice). Multiple phenuiviruses, such as Dabie bandavirus, Rift Valley fever phlebovirus, and Rice stripe tenuivirus, are highly pathogenic to humans, animals, or plants. They impose heavy global burdens on human health, livestock industry, and agriculture and are research hotspots. In recent years the taxonomy of Phenuiviridae has been expanded greatly, and researches on phenuiviruses have made significant progress. With these advances, this review drew a novel panorama regarding the biomedical significance, distribution, morphology, genomics, taxonomy, evolution, replication, transmission, pathogenesis, and control of phenuiviruses, to aid researchers in various fields to recognize this highly adaptive and very important virus family.

The origin of the nucleus remains a great mystery in life science, although nearly two centuries have passed since the discovery of nuclei. To date, studies of eukaryogenesis have focused largely on micro-evolutionary explanations. Here, we examined macro-patterns of C-values (the total amount of DNA within the haploid chromosome set of an organism) for over 110,000 species and the chromosome numbers for over 11,000 species and their potential links with the state of atmospheric oxidation over geological time. Eukaryogenesis was in sync with an over 2.5 order-of-magnitude increase in genome size from prokaryote to eukaryote, and also with a rapid rise of atmospheric oxidation, suggesting that eukaryogenesis would have resulted from a regime shift of genomes driven by the oxidation-driven complexification and structuralization (e.g. chromatin packing).

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. It also has been widely used to study structural variants, phase haplotypes and more. Here, we introduce the assembler— SMARTdenovo, which is an SMS assembler that follows the overlap-layout-consensus (OLC) paradigm. SMARTdenovo (RRID: SCR_017622) was designed to be a fast assembler that did not require highly accurate raw reads for error correction, unlike other, contemporaneous SMS assemblers. It has performed well for evaluating congeneric assemblers and has been successful for a variety of assembly projects. It is compatible with Canu for assembling high-quality genomes, and several of the assembly strategies in this program have been incorporated into subsequent popular assemblers. The assembler has been in use since 2015, and here we provide information on the development of SMARTdenovo and how to implement its algorithms into current projects.

Background: Transposable elements (TEs) constitute the vast majority of all eukaryotic DNA, and display extreme diversity, with thousands of families. Given their abundance and diversity, TEs discovery and annotation becomes challengeable. At present, tools and databases have built libraries to mask TEs in genomes based on de novo- and homology-based identification strategies, but no consensus criteria about which tools should be used have been proposed. Results: In the de novo-based strategy, we compared performances of TE libraries developed by four commonly used tools, including RepeatModeler, LTR_FINDER, LTRharvest, and MITE_Hunter, by using a simulated genome as a standard control. The results showed that the performance of RepeatModeler decreased as it was combined with either LTR_FINDER or LTRharvest. Combination of RepeatModeler and MITE_Hunter showed better performance than RepeatModeler and MITE_Hunter alone. In the homology-based strategy, we evaluated different sources from a taxonomic point of view to build an accurate TE library. When we selected a library from databases to identify TEs for Arabidopsis thaliana genome, the library from a genus genetically closer to Arabidopsis achieved better performance than other genera with further genetic distance. Without the Arabidopsis, combination of top three genera closer to Arabidopsis showed better performance than combination of all genera. Conclusion: This study proposes a series of recommendations to perform an accurate TE annotation: 1) For de novo-based strategy, RepeatModeler and MITE_Hunter are suggested to build a TE library; 2) For homology-based strategy, it is recommended to use library of genus genetically close to the species rather than use combined library from all genera.

Heteromorphic sex chromosomes are rarely found in plants. They were observed only in 47 species from phylogenetically distant families, suggesting that the evolution of sex chromosomes was independent in these species. It was shown that DNA repeat sequences are one of the major factors driving sex chromosomes evolution, and an accumulation or elimination of the repetitive DNA elements are closely linked with the formation of differences in the sex chromosomes. The goal of this study was to characterize the transposon composition in male and female plants of Cannabis sativa L., Humulus lupulus L. and Humulus japonicus Siebold &amp; Zucc. For the first time, the male and female genomes of H. japonicus as well as male genomes of H. lupulus and C. sativa have been sequenced (there were no open data about them). The analysis of genome-wide sequencing data with using Repeatexplorer2 and author’s scripts was carried out. It was shown that accumulation of Ty3-gypsy may be associated with speciation in Cannabaceae family which is the opposite of the theory of speciation throw whole-genome duplication. Moreover, the sex-specific DNA repeat clusters in C. sativa and H. japonicus were found. The analysis also revealed that the concentration of Tekay, Retand and Ikeros repeats in the Y chromosome of C. sativa is lower than in the X chromosome and the Angela concentration is higher in the Y chromosome.

Combined awareness about the power and limitations of bioinformatics and molecular biology enables advanced research based on high-throughput data. Despite an increasing demand for scientists with a combined background in both fields, the education in dry lab and wet lab is often separated. This work describes an example of integrated education with focus on genomics and transcriptomics. Participants learn computational and molecular biology methods in the same practical course. Peer-review is applied as a teaching method to foster cooperative learning of students with heterogeneous backgrounds. Evaluation results indicate acceptance and appreciation of this approach.

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole cell to acellular pertussis vaccines, although other factors may also contribute. To develop future vaccines and improve current vaccination strategies, it is critical to understand factors influencing the generation of immunological memory. We applied high-dimensional flow cytometry to investigate changes in B cells in individuals of different ages and distinct priming backgrounds upon administration of an acellular pertussis booster vaccine. These findings were correlated to vaccine-specific plasma cells and serum Ig levels. Expansion and maturation of plasma cells 7 days post-vaccination was the most prominent cellular change in all age groups, and was most pronounced for more mature IgG1+ plasma cells. Cellular responses were stronger in individuals primed with whole cell vaccine than in individuals primed with acellular vaccine. Moreover, IgG1+ plasma cell expansion weakly correlated with Prn- and PT- specific serum IgG levels. Our study points at plasma cells as a potential early cellular marker of an immune response and contributes to understanding differences in immune responses between age groups and priming backgrounds.

Germline mutations in predisposition genes account for only 20% of all familial colorectal cancer (CRC) and the remaining genetic burden may be due to rare high-to-moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants; a coding variant in APC down-regulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5’ untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additional APCDD1 variants in a cohort of 1705 familial CRC patients and no further HDAC5 variants. Proliferation assays indicated an insignificant proliferative impact for the APCDD1 variant. Luciferase reporter assays using the HDAC5 variant resulted in an enhanced promoter activity. Targeting of transcription factor binding sites of SNAI-2 and TCF4 interrupted by HDAC5 variant showed a significant impact of TCF4 on promoter activity of mutated HDAC5. Our findings contribute not only to the identification of unrecognized genetic causes of familial CRC but also underline the importance of 5´UTR variants affecting transcriptional regulation and the pathogenesis of complex disorders.

The KARS gene encodes the aminoacyl-tRNA synthetase (aaRS) which activates and joins the lysin with its corresponding transfer RNA (tRNA), through the ATP-dependent aminoacylation of the amino acid. The KARS gene mutations have been linked to diverse neurologic phenotypes such as: neurosensorial hearing loss, leukodistrophy, microcephaly, developmental delay or regression, peripheral neuropathy, cardiomyopathy, impairment of the mitochondrial respiratory chain, hyperlactatemia, among others. This article presents the case of a Colombian pediatric patient with two pathological missense variants in a compound heterozygous state in the KARS gene.

A possible way to valorize citrus peels, which are byproducts of the juice extraction industry, is to use them as natural biopreservatives. In this paper we present early results from a compared Solvent Free Microwave Extraction (SFME) with Hydro-Distillation (HD) and Cold Pressing (CP) of essential oils (EOs) using fresh orange peel (Citrus sinensis L. var. Valencia late), a by-product in the production of orange juice in Algeria. The EOs were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). All extracted C. sinensis EOs were chemotype limonene (94.64 to 95.48%). SFME is performed without added any solvent or water. SFME increases EO yield and eliminate wastewater treatment, resulting in a great progress in terms of time and cost efficiency. In its second part, the present study was conducted to evaluate “in vitro”, the antioxidant activities of Solvent Free Microwave (SFM) extracted orange EO by using the DPPH• (2,2-di-phenyl-1-picrilhydrazyl) free radical scavenging assay. The ability of orange EO to scavenge the free radical DPPH• was high, exceeding 80%. The result of the DPPH assay gives an IC₅₀ range value of 89.25 μg/mL (0.09 mg/mL) for the studied sample. Accordingly to the scientific literature, C. sinensis EO tested in the present study presented strong antioxidant activity, when looking to its values of AAI = 1.12 μg/mL. The feasibility of biopreservation used EOs as an alternative to synthetic techniques for liquid whole egg (LWE) stored under commercial retail conditions was investigated. The orange EO extracted by SFM was screened for its antibacterial and antioxidant activities in LWE at concentrations of 0.1, 0.3 and 0.5%. The TBA-RS results showed that the EO treatments significantly (p < 0.05) reduced the lipid oxidation in LWE. The long term oxidative, microbial and organoleptical stability of the LWE during display was positively influenced by orange EO treatments. Therefore, the results obtained here confirm that EO treatment as a promising technology to extend the commercial shelf-life of liquid egg products during retail/display.

Poplar and willow species in the Salicaceae are dioecious, yet have been shown to use different sex determination systems located on different chromosomes. Willows in the section Vetrix are interesting for comparative studies of sex determination systems, yet genomic resources for these species are still quite limited. Only a few annotated reference genome assemblies are available, despite many species in use in breeding programs. Here we present de novo assemblies and annotations of 11 shrub willow genomes from six species. Copy number variation of candidate sex determination genes within each genome was characterized and revealed remarkable differences in putative master regulator gene duplication and deletion. We also analyzed copy number and expression of candidate genes involved in floral secondary metabolism, and identified substantial variation across genotypes, which can be used for parental selection in breeding programs. Lastly, we report on a genotype that produces only female descendants and identified gene presence/absence variation in the mitochondrial genome that may be responsible for this unusual inheritance.

Background. Mitochondrial dysfunction has been identified as a pathophysiological hallmark of disease onset and progression in patients with Parkinsonian disorders. Besides the overall emergence of gene therapies in treating these patients, this highly relevant molecular concept has not yet been defined as a target for gene therapeutic approaches. Methods. This narrative review will discuss the experimental evidence suggesting mitochondrial dysfunction as a viable treatment target in patients with monogenic and idiopathic Parkinson’s disease. In addition, we will focus on general treatment strategies and crucial challenges which need to be overcome. Results. Our current understanding of mitochondrial biology in parkinsonian disorders opens up the avenue for viable treatment strategies in Parkinsonian disorders. Insights can be obtained from primary mitochondrial diseases. However, substantial knowledge gaps and unique challenges of mitochondria-targeted gene therapies need to be addressed to provide innovative treatments in the future. Conclusions. Mitochondria-targeted gene therapies are a potential strategy to improve an important primary disease mechanism in Parkinsonian disorders. However, further studies are needed to address the unique design challenges for mitochondria-targeted gene therapies.

Choanoflagellates, the closest living relatives of animals, have the potential to reveal the genetic and cell biological foundations of complex multicellular development in animals. Here we describe the history of research on the choanoflagellate Salpingoeca rosetta. From its original isolation in 2000 to the establishment of CRISPR-mediated genome editing in 2020, S. rosetta provides an instructive case study in the establishment of a new model organism.

The development of CRISPR associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly and quantitatively monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses at the end of study. Here we report a new CRISPR “footprinting” approach to activate luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior Cre-detector system and is designed for Cas editors able to target LoxP including gRNAs including SaCas9 and ErCas12a [1, 2]. These CRISPRs cut specifically within LoxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living Cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to Cre recombinase demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated LoxP animal models.

Entamoeba histolytica, like other Organismes, is characterized by diversity and heterogeneity in its genetic content, which is one of the most important reasons for survival, and the increase in susceptibility to infection.Non-condensation of chromosomes during the process of cell division and the ambiguity of the chromosomal ploidy makes predicting the exact chromosomal number difficult. Genes distributed across 14 chromosomes as well as many extra-chromosome elements. Most Genes composed of one axon only, with Introns in 25% of Genes. This genome is characterized by the presence of Polymorphic internal repeat regions, and several gene families, one of these large families encoding Transmembrane kinas, Cysteine protease (CP), SREHP protein, and others.

The constitutively active tyrosine kinase BCR/ABL1 oncogene plays a key role in human chronic myeloid leukemia development and disease maintenance, and determines most of the features of this leukemia. For this reason, tyrosine kinase inhibitors are the first-line treatment, offering most patients a life expectancy like that of an equivalent healthy person. However, since the oncogene is not destroyed, lifelong oral medication is essential, even though this trigger adverse effects in many patients. Furthermore, leukemic stem cells remain quiescent and resistance is observed in approximately 25% of patients. Thus, new therapeutic alternatives are still needed. In this scenario, the emergence of CRISPR technology can offer a definitive treatment based on its capacity to disrupt coding sequences. This review describes CML disease and the main advances in the genome-editing field by which it may be treated in the future.

Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with diverse chemical structures and potent biological activities and toxicities. The chemical identification of these compounds remains a major bottleneck. Strategies that can prioritize the most prolific strains and novel compounds are of great interest. Here, we combine chemical analysis and genomics to investigate the chemodiversity of secondary metabolites based on their pattern of distribution within some cyanobacteria. Planktothrix being a cyanobacterial genus known to form blooms worldwide and to produce a broad spectrum of toxins and other bioactive compounds, we applied this combined approach on four closely related strains of Planktothrix. The chemical diversity of the metabolites produced by the four strains was evaluated using an untargeted metabolomics strategy with high-resolution LC-MS. Metabolite profiles were correlated with the potential of metabolite production identified by genomics for the different strains. Although, the Planktothrix strains present a global similarity in term biosynthetic cluster gene for microcystin, aeruginosin and prenylagaramide for example, we found remarkable strain-specific chemo-diversity. Only few of the chemical features were common to the four studied strains. Additionally, the MS/MS data were analyzed using Global Natural Products Social Molecular Networking (GNPS) to identify molecular families of the same biosynthetic origin. In conclusion, we present an efficient integrative strategy for elucidating the chemical diversity of a given genus and link the data obtained from analytical chemistry to biosynthetic genes of cyanobacteria.

The survival analysis of the Cancer Genome Atlas (TCGA) dataset is a well-known method to discover the gene expression-based prognostic biomarkers of head and neck squamous cell carcinoma (HNSCC). A cutoff point is usually used in survival analysis for the patients' dichotomization in the continuous gene expression. There is some optimization software for cutoff determination. However, the software's predetermined cutoffs are usually set at the median or quantiles of gene expression value to perform the analyses. There are also few clinicopathological features available on their pre-processed data sets. We applied an in-house workflow, including data retrieving and pre-processing, feature selection, sliding-window cutoff selection, Kaplan-Meier survival analysis, and Cox proportional hazard modeling for biomarker discovery. In our approach for the TCGA HNSCC cohort, we scanned human protein-coding genes to find optimal cutoff values. After adjustment with confounders, the clinical tumor stage and the surgical margin involvement are independent risk factors for patients' prognosis. According to the resulting tables with Bonferroni-adjusted P value under the optimal cutoff and the hazard ratio, three biomarker candidates, CAMK2N1, CALML5, and FCGBP, are significantly associated with the patients' overall survival. We validated this discovery by using the other independent HNSCC dataset (GSE65858). Thus, we suggest the transcriptomic analysis could help for biomarker discovery.

Urinary cytology is a useful, essential diagnostic method in routine urological clinical practice. Liquid-based cytology (LBC) for urothelial carcinoma screening is commonly used in the routine clinical cytodiagnosis because of its high cell collection rate. Since conventional screening processes by cytoscreeners and cytopathologists using microscopes is limited in terms of human resources, it is important to integrate new deep learning methods that can automatically and rapidly diagnose a large amount of specimens without delay. The goal of this study was to investigate the use of deep learning models for the classification of urine LBC whole-slide images (WSIs) into neoplastic and non-neoplastic (negative). We trained deep learning models using 786 WSIs by transfer learning, fully supervised, and weakly supervised learning approaches. We evaluated the trained models on two test sets (equal and clinical balance) with a combined total of 750 WSIs, achieving ROC-AUCs for WSI diagnosis in the range of 0.984-0.990 by the best model, demonstrating the promising potential use of our model for aiding urine cytodiagnostic processes.

Amino acid efflux and influx transport systems play vital roles in industrial microorganisms’ cell growth and metabolism. However, although biochemically characterized, most amino acid transporters remain unknown at the molecular level in Bacillus licheniformis. This study focuses on the molecular and functional characterizations of three transporters, YdgF, YvbW, and YveA, mainly when catalyzing the cross-membrane flux of L-Aspartate. When growing in the minimal medium with L-Asp as the only carbon and nitrogen source, the growth of strains lacking proteins YdgF, YvbW, and YveA was significantly inhibited compared with wild-type strains, while supplementing the expression of the corresponding proteins in the single-gene knockout strains can alleviate the inhibition to some extent. Upon overexpression, the recombinant proteins mediate the accumulation of L-aspartate to varying degrees. Compared with wild-type strains, the single knockout strains of the three protein genes exhibited reduced absorption of L-aspartate. In addition, this paper focuses on the effects of these three proteins on the absorption of β-alanine, L-glutamate, D-serine, D-alanine, and glycine.

The use of whole-body vibration (WBV) for therapeutic purposes is far from being standardized and only very recently an empirical foundation for reporting guidelines for human WBV studies has been published. Controversies about safety and therapeutic dosage stll exist. The present study aimed to investigate the metabolic and mechanical effects of low-intensity WBV in according to the ISO 2631 norm on subjects with obesity. 41 obese subjects (BMI≥ 35 kg/mˆ2) were recruited to participate in a 3-week multidisciplinary inpatient rehabilitation program including fitness training and WBV training. During WBV the posture was monitored with an optoelectronic system with 6 infrared cameras (Vicon, Vicon Motion System, Oxford, UK). The primary endpoints were: variation in body composition, factors of the metabolic syndrome, functional activity (sit-to-stand and 6-min walking test), muscle strength, and quality of life. Secondary endpoints were: modification of irisin, testosterone, growth hormone, IGF1 levels. We observed significant changes in salivary irisin levels, Group 2 (p&lt;0.01) as compared to the control group, while muscle strength, function, and other metabolic and hormonal factors did not change after a 3-week low-intensity WBV training respect control group. Future studies are needed to deeper investigate the potential metabolic effect of low-intensity WBV in managing weight.

Stereotactic radiosurgery (SRS) has become increasingly important in the management of brain metastases due to improving systemic disease control and rising incidence. Initial trials demonstrated SRS with whole brain radiotherapy (WBRT) improved local control rates versus WBRT alone. Concerns with WBRT associated neurocognitive toxicity have contributed to greater use of SRS alone, including for patients with multiple metastases and following surgical resection. Molecular information, targeted agents and immunotherapy have also altered the landscape for the management of brain metastases. This review summarises current and emerging data on the role of SRS in the management of brain metastases.

Due to the global warming and dynamic changes in pathogenic virulence, leaf rust caused by Puccinia triticina has greatly expanded its epidermic region and become a severe threat to global wheat production. Genetic bases of wheat resistance to leaf rust mainly relies on the leaf rust resistance (Lr) gene or quantitative trait locus (QLr). Although these genetic loci have been insensitively studied during the last two decades, an updated overview of Lr/QLr in a genome-wide level is urgently needed. This review summarized recent progresses in genetic studies of wheat resistance to leaf rust. Wheat germplasms with great potentials in genetic improvement of resistance to leaf rust were highlighted. Key information about the genetic loci carrying Lr/QLr were summarized. A genome-wide chromosome distribution map for all the Lr/QLr was generated based on the released wheat reference genome. In conclusion, this review has provided valuable sources for both wheat breeders and researchers to understand the genetics of resistance to leaf rust in wheat.

Plant diseases are among the major factors affecting plant productivity. Biological control of plant diseases is preferred over chemical control as it is environment-friendly, cost-effective, and sustainable. Among many microbes capable of providing biological control of plant diseases, probiotic Bacillus species are most promising as they can survive in adverse conditions, provide plants with a wide range of benefits including protection from phytopathogens. Wheat blast caused by Magnaporthe oryzae Triticum pathotype (MoT) has emerged as a potential threat to global wheat production. Due to unreliability of fungicides and limited cultivar resistance, we aimed to screen and identify potential antagonist bacteria collected from internal tissues of rice and wheat seeds to determine their in vitro and in vivo inhibitory effects against MoT. Dual culture and seedling assays were performed to evaluate the efficacy of probiotic bacteria. Out of 170 bacterial isolates, three bacteria (BTS-3, BTS-4, and BTLK6A) were screened as potential antagonists against MoT in vitro. Artificial inoculation at the seedling stage showed that the isolates BTS-4, BTS–3, and BTLK6A reduced 89, 88, and 85% of wheat blast disease severity, respectively, compared to mock-inoculated control. The bacterial isolates were identified as Bacillus subtilis (BTS-3) and B. velezensis (BTS-4 and BTLK6A) through genome phylogeny. The whole genome sequence of these three bacterial strains decoded a number of orthologs to intrinsic genes of antimicrobial peptides, antioxidant defense enzymes, cell wall degrading enzymes, compounds involved in the induction of systemic resistance (ISR) in host plants, and volatile compounds to make them promising biologicals to control MoT in wheat. Combined data of in vitro and in vivo along with genome analysis suggest that Bacillus spp. suppress the destructive wheat blast disease likely through antibiosis and ISR in the host plants. Further field evaluation and characterization of antimicrobial compounds are needed for a better understanding of the mode of action and practical recommendation of these bacteria for wheat blast control in the farmers’ fields.

Human cancers comprise an heterogeneous array of diseases with different progression patterns and responses to therapy. However, they all develop within a host context that constraints their natural history. As it occurs with the diversity of organisms, one can conjecture that there is order in the cancer multiverse. Is there a way to capture the broad range of tumor types within a space of the possible? Here we define the oncospace, a coordinate system that integrates the ecological, evolutionary and developmental components of cancer complexity. The spatial position of a tumor results from its departure from the healthy tissue along these three axes, and progression trajectories inform about the components driving malignancy across cancer subtypes. We postulate that the oncospace topology encodes new information regarding tumorigenic pathways, subtype prognosis and therapeutic opportunities: treatment design could benefit from considering how to nudge tumors towards empty evolutionary deserts in the oncospace.

Global crop yields are estimated to be reduced by 30–40% per year on account of plant pests and pathogens. Agricultural insect pests raise concerns about constraining global food security and climate changes contributing to the rise of infestation. The current management relies on plant breeding, associated or not with transgenes and chemical pesticides. Both approaches face serious technology obsolescence on the field due to resistance breakdown or development of insecticide resistance. The need for new Modes of Action (MoA) approaches in managing crop health grows each year, driven by market demands to reduce economic losses and phytosanitary requirements to meet the consumer perception. Disabling pest genes by sequence-specific expression silencing is considered a promising tool in the development of environment and health respectful biopesticides. The specificity conferred by long dsRNA-base solutions give support to minimizing effects on off-targeted genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the current status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera by the employment of non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to imminent innovative solutions. Considerations about the regulamentation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives reveal a worthy effort to accomplish controlling Lepidoptera pests with this new mode of action to provide more sustainable and reliable technologies to field management. New data on genomics of this taxon encourage the increment of a customized target genes portfolio. As a case of study, we illustrate how dsRNA and associated methodologies could be applied to control an important Lepidopteran coffee pest.

Covid-19 has caused infections and deaths worldwide. While research in the field of Data Science has contributed good predictions of positive Covid-19 case numbers, this study's review of literature shows there is little research in the use of variants of the virus in predictions. We set out to define and evaluate novel variant features. We find that features relating to variant trends, thresholds and amino acid substitutions are especially powerful in two tasks. In the first task, predicting Covid-19 case numbers, accuracy improved from 71.53% without variant features to 82.12% with variant features. In the second task, predicting transmission severity of variants between two classes, we created a method to build some variable ensembles through selecting appropriate models that are generated with variant features. The test results showed that our ensembles are more accurate and reliable. One particular ensemble of 14 models correctly classified 90.91% of variants, outperforming other models including the popular Random Forest ensemble. In addition, as the variant features have represented more underlying information about Covid-19 pathophysiology, our ensemble methods use only a few data samples to achieve an accurate prediction. The ensemble of 14 models uses only 50 cases of each variant, an ability that could be exploited for early detection of highly infectious variants. These research findings may benefit public health professionals, policy makers, and the research community in the collective efforts to overcome this disease.

Plastid genomes are in general highly conserved given their slow evolutionary rate, thus large changes in their structure are unusual. However, when specific rearrangements are present, they are often phylogenetically informative. Asteraceae is a highly diverse family whose evolution is long driven by polyploidy (up to 48x) and hybridisation, both processes usually complicating systematic inferences. In this study, we have generated one of the most comprehensive plastome-based phylogenies of family Asteraceae, providing information about the structure, genetic diversity, and repeat composition of these sequences. By comparing the whole plastome sequences obtained, we confirmed the double inversion located in the long single copy region, for most of the species analysed (with the exception of basal tribes), a well-known feature for Asteraceae plastomes. We also show that genome size, gene order and gene content are highly conserved along the family. However, species representative of the basal subfamily Barnadesioideae -as well as in the sister family Calyceraceae - are lacking the pseudogene rps19 located in one inverted repeat. The phylogenomic analysis conducted here, based on 63 protein-coding genes, 30 transfer RNA genes and 21 ribosomal RNA genes from 36 species of Asteraceae, are overall consistent with the general consensus for the family’s phylogeny, while resolving the position of tribe Senecioneae and revealing some incongruences at tribe level between reconstructions based on nuclear and plastid DNA data.

Marseille-Q4369 is a strain that we isolated from human healthy skin and characterized by taxono-genomic approach. Marseille-Q4369 exhibited 99.80% 16S rRNA sequence similarity with Agrococcus pavilionensisT the phylogenetically closest bacterium with standing in nomenclature. Furthermore, digital DNA–DNA hybridization revealed a maximum identity similarity of only 52.4% and an OrthoANI parameter provided a value of 93.63% between the novel organism and Agrococcus pavilionensisT. Marseille-Q4369 was observed to be a yellowish-pigmented, Gram-positive, coccoïd, facultative aerobic bacterium, and belonging to the Microbacteriaceae family. The major fatty acids detected are 12-methyl-tetradecanoic acid (66%), 14-methyl-hexadecanoic acid (24%) followed by 13-methyl-tetradecanoic acid (5%). The genome size of strain Marseille-Q4369 was 2,737,735-bp long with a 72,27 % G+C content. Taken altogether, these results confirm the status of this strain as a new member of the Agrococcus genus for which the name of Agrococcus massiliensis is proposed (=CSUR-Q4369 = DSM112404).

Influenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal influenza vaccination is considered the most efficient strategy to prevent and control IBV infections. Live attenuated influenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness have recently come into question. Thus, the opportunity exists to find alternative approaches to improve overall influenza vaccine effectiveness. Two alternative IBV backbones were developed with re-arranged genomes, re-arranged M (FluB-RAM) and a re-arranged NS (FluB-RANS). Both re-arranged viruses showed temperature sensitivity in vitro compared to the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both re-arranged viruses induced antibodies against HA with hemagglutination inhibition titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efficacious LAIV candidates to protect mice against IBV.

Asaia bacteria commonly comprise part of the microbiome of many mosquito species in the genera Anopheles and Aedes, including important vectors of infectious agents. Their close association with multiple organs and tissues of their mosquito hosts enhances the potential for paratransgenesis for delivery of anti-malaria or anti-virus effectors. The molecular mechanisms involved in the interactions between Asaia and mosquito hosts, as well as Asaia and other bacterial members of the mosquito microbiome, remained unexplored. Here, we determined the genome sequence of the strain W12 isolated from Anopheles stephensi mosquitoes, compared them to other Asaia species associated with plants or insects, and investigated some properties of the bacteria relevant to their symbiosis with host mosquitoes. The assembled genome of strain W12 has a size of 3.94 MB, which is the largest among Asaia spp studied so far. At least 3,585 coding sequences were predicted. The insect-associated Asaia including strain W12 carried more glycoside hydrolase (GH) encoding genes (31 per genome) than those isolated from plants (22 per genome). W12 had the most predicted regulatory protein components (213) among the selected Asaia (ranging from 131 to 211), indicating its great capability to adapt to frequent environmental changes in the mosquito gut. Two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases (cyoABCD-1 and cyoABCD-2) were found in most of Asaia genomes, which possibly offer alternative terminal oxidases and allow the flexible transition of respiratory pathways. Genes involved in the production of acetoin and 2,3-butandiol have been identified in Asaia sp. W12.

We are in a new chapter of crop and livestock improvement with the emergence of genome editing. This latest generation of molecular tools can be used to make targeted changes in a genome including insertions, deletions, and mutations. With new advances comes new risks for unintended changes and impacts, thus the need for appropriate risk assessment for product development and to inform regulatory measures. Though CRISPR/Cas has arisen as the predominant technology, there are multiple types of genome editing tools each with pros and cons depending on the organism and desired outcome. Furthermore, each editing tool differs in specificity as they may edit non-intended sites, referred to as off-target edits. The consensus of the agricultural editing community is to avoid off-target editing through design and detection, instead of determining whether off-target editing in each case is detrimental. The design of a targeting component, the tool chosen, and the identification of the edit(s) made are the critical factors in avoiding off-target edits and confirming intended edits in final products that are released commercially. The limited amount of head-to-head comparisons of genome editing tools in diverse crops and livestock make it difficult to develop broad conclusions and best practices, which is further compounded by the diversity of techniques, targets, and processes. Developers and breeders should consult the literature and test as needed to determine which editing technology will be the most effective for their purposes, especially as more tools with altered efficiency and specificity become available. Yet, the lack of off-target edits in studies that employed careful design of targeting components followed by wide testing for on- and off-target edits bodes well for the use of genome editing with proper precautions of target selection and screening.

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimise genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (Rapid Evolution Detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with haploid progeny of industrial yeast strains to produce stress tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate optimal SCRaMbLE induction times of different Cre-recombinse expression systems for the development of industrial strains.

In the present study, the complete mitochondrial genome of the New Zealand parasitic blowfly Lucilia sericata (green bottle blowfly) field strain NZ_LucSer_NP was generated using next-generation sequencing technology. The length of complete the mitochondrial genome is 15,938 bp, with 39.4% A, 13.0% C, 9.3% G, and 38.2% T nucleotide distribution. The complete mitochondrial genome consists of 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and a and a 1,124 bp non-coding region, similar to most metazoan mitochondrial genomes. Phylogenetic analysis showed that L. sericata NZ_LucSer_NP forms a monophyletic cluster with the remaining six Lucilia species and the Calliphoridae are polyphyletic. This study provides the first complete mitochondrial genome sequence for a L. sericata blowfly species derived from New Zealand to facilitate species identification and phylogenetic analysis.