A recent manuscript (Zhou, P. et al. “A pneumonia outbreak associated with a new coronavirus of probable bat origin”, Nature 579, 270–273 (2020). https://doi.org/10.1038/s41586-020-2012-7) from Wuhan Institute of Virology claimed the identification of a bat coronavirus, RaTG13, which showed 96.2% genome homology with SARS-CoV-2. In this paper, we raise the puzzling observations surrounding the identification, characterization, unique genome features of this RaTG13 strain, as well as its 100% nucleotide identity in partial RdRp gene with another bat coronavirus strain BtCoV/4991. And the paper presented premature hypothesis of potential bat origin of SARS-CoV-2 while RaTG13 strain was not successfully isolated. We also present the concerns on the methodology, data quality and experiment procedures described in this paper. We call for the authors to provide additional data, to share related samples to be verified and further characterized by other scientists.

SARS-CoV-2 is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive-sense single-stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA-dependent-RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that Vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

Starting from December 2019, coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures and supporting apparatuses. Remdesivir was reported by World Health Organization (WHO) as the most promising drug currently available for the treatment of COVID-19. Here, we use molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). In the absence of a crystal structure of the SARS-CoV-2 RdRp, we first construct the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identify of 95.8%). We then build the putative binding mode by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp. The putative binding structure is further optimized with molecular dynamics simulations and demonstrated to be stable, indicating a reasonable binding mode for remdesivir. The relative binding free energy of remdesivir is calculated to be -8.28 ± 0.65 kcal/mol, much stronger than the natural substrate ATP (-4.14 ± 0.89 kcal/mol) which is needed for the polymerization. The ~800-fold improvement in the K_d from remdesivir over ATP indicates an effective replacement of APT in blocking of the RdRp binding pocket. Key residues D618, S549 and R555 are found to be the contributors to the binding affinity of remdesivir. These findings demonstrate that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA reproduction, with key residues also identified for future lead optimization and/or drug resistance studies.

The existence of a common ancestor to all living organisms in Earth is a necessary corollary of Darwin idea of common ancestry. The Last Universal Common Ancestor (LUCA) has been normally considered as the ancestor of cellular organisms that originated the three domains of life: Bacteria, Archaea and Eukarya. Recent studies about the nature of LUCA indicate that this first organism should present hundreds of genes and a complex metabolism. Trying to bring another of Darwin ideas into the origins of life discussion, we went back into the prebiotic chemistry trying to understand how LUCA could be originated under gradualist assumptions. Along this line of reasoning, it became clear to us that the definition of another ancestral should be of particular relevance to the understanding about the emergence of biological systems. Together with the view of biology as a language for chemical translation, on which proteins are encoded into nucleic acids polymers, we glimpse a point in the deep past on which this Translation mechanism could have taken place. Thus, we propose the emergence of this process shared by all biological systems as a point of interest and propose the existence of this pre-cellular entity named FUCA, as the First Universal Common Ancestor. FUCA was born in the very instant on which RNA-world replicators started to be capable to catalyze the bonding of amino acids into oligopeptides. FUCA has been considered mature when the translation system apparatus has been assembled together with the establishment of a primeval, possibly error-prone genetic code. This is FUCA, the earliest ancestor of LUCA’s lineage.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding, and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are freely available online, either through web applications or public code repositories.

COVID-19 has emerged as deadly pandemic worldwide with no vaccine or suitable antiviral drugs to prevent or cure the disease. Because of the time-consuming process to develop new vaccines or antiviral agents, there has been a growing interest in repurposing some existing drugs to combat SARS-CoV-2. Vitamin D is known to be protective against acute respiratory distress syndrome (ARDS), pneumonia and cytokine storm. Recently it has been used as a repurposed drug for the treatment of H5N1 virus-induced lung injury. Circumstantial evidences indicate that people with low level of vitamin D are more susceptible to SARS-CoV-2. Although, vitamin D was suggested to interfere with viral replication, its interaction with any SARS-CoV-2 protein is unexplored yet. Beside this, ivermectin, a well-known anti-parasitic agent, exhibits potent anti-viral activities in vitro against viruses such as HIV-1 and dengue. Very recently, ivermectin has been found to reduce viral load of SARS-CoV-2 in vitro. We have analyzed available structures of SARS-CoV-2 proteins to identify probable binding partner(s) of vitamin D and ivermectin through knowledge-based docking studies and figured out possible implication of their binding in SARS-CoV-2 infection. Our observations suggest that the non-structural protein nsp7 possesses a potential site to house 25-hydroxyvitamin D3 (VDY) or the active form of Vitamin D, calcitrol. Binding of vitamin D with nsp7 likely to hamper the formation of nsp7-nsp8 complex which is required to bind with RNA dependent RNA polymerase (RdRP), nsp12 for optimal function. On the other hand, potential binding site of ivermectin has been identified in the S2 subunit of trimeric spike(S) glycoprotein of SARS-CoV-2. We propose that deeply inserted mode of ivermectin binding at three inter-subunit junctions may restrict large scale conformational changes of S2 helices which is necessary for efficient fusion of viral and host membrane. Our study, therefore, opens up avenues for further investigations to consider vitamin D and ivermectin as potential drugs against SARS-CoV-2.

Emergence of zoonotic-human pathogens is proven to be a lethal threat to public health, and RNA virus including influenza viruses, severe acute respiratory syndrome coronavirus, middle east respiratory syndrome coronavirus, Wuhan coronavirus (COVID-19), plays a pivotal role. As those viruses as airborne microorganisms spread mainly by tiny airborne particles, it is important to de-active those airborne particles before their entry into human bodies. In this study, we investigated the effect of far infrared (FIR) radiation on inhibition of airborne microorganisms. The result confirmed that double stand DNA from airborne microorganisms were stable under mild FIR radiation. However, single strand RNA from them was found to be sensitive to FIR radiation, indicating that RNA virus in airborne particles is instable under FIR radiation. Based on this observation, two models on usage of FIR radiation to prevent RNA virus transmission and cure RNA virus infection were proposed, implying that FIR radiation might be a cheap, convenient, and efficient method in clinic to treat RNA virus.

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in China towards the end of 2019, and has spread rapidly ever since. Previous studies showed that some virus could affect the reproductive system and cause long-term complications. Recent studies exploring the source of SARS-CoV-2 using genomic sequencing have revealed that SARS-CoV-2 enters the host cells via the angiotensin-converting enzyme II (ACE2), the receptor that recognizes SARS-CoV. To investigate the expression of ACE2 and to explore the potential risk of infection in the reproductive system, we performed a thorough bioinformatic analysis on data from public databases involving RNA expression, protein expression, and single-cell RNA expression studies. The analyzed data showed high levels of ACE2 mRNA and protein expression in the testis and spermatids and equal levels of ACE2 expression in the uterus and lung. Comprehensive single-cell analysis identified ACE2 expression in the lung, testis, spermatids, and uterus. In conclusion, this study revealed the potential risk associated with the SARS-CoV-2 infection in the reproductive system and predicted that long-term complications might have a significant impact on the prevention and management of COVID-19, the disease caused upon infection with SARS-CoV-2.

Background: Gut-Brain-Axis provides bidirectional communicational route; imbalance of which can have pathophysiological consequences. It is a frontier in autism research, affects 85% of autistic children (NIH report). Their microbiome has few overall microbes and smaller number of health promising microbes than their neurotypical peers. We hypothesize autism gut might play a role in manifestation of autism behaviours and on treatment, can revert back to normal behaviour considerably. The aim is to better understand to what degree gut microbiota of autism subjects differs from controls and identify bacterial species present exclusively in autism. Materials and Methods: 16s-rRNA-sequence of autism-subjects were retrieved from the American Gut Project Archive. Taxonomic assignment was inferred by similarity based methods using Quantitative Insights Into Microbial Ecology (QIIME). Species abundance was characterized and co-occurrence network was built to infer species interaction using measures of diversity. Statistical parameters were considered to validate the findings. Result: A total of 206 (1.8%) of American Gut Project datasets onstituted of autistic samples. Various bacteria such as Akkermansia sp., and Prevotella sp., were harboured in higher abundance in autistic children with statistical significance than in controls. Conclusion: These findings indicate connecting-link between gut-microbiome-brain-axis and autistic behaviour which can result in improved management

Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The eCDC and the WHO recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the PCR-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor a possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490-509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariant when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.1 NTD and a BA.2 RBD, which can only be detected by using specially designed PCR primers. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.

RaTG13 (a bat derived SARS-like CoV) is the closest relative sequence of SARS-CoV-2 reported till date. The sample from which RaTG13 was sequenced was a bat fecal swab collected in 2013 from Tongguan, Mojiang, Yunnan province, China. The Illumina based sequence of RaTG13, MN996532.1, was deposited on 27^th Jan 2020 and the raw data (Illumina), https://www.ncbi.nlm.nih.gov/sra/SRX7724752[accn]. There are discrepancies in dates about when the metagenome sequencing of RaTG13 sample was done (2018 or 2020), both stated by the same corresponding author. Comparison of the RNA Seq data of RaTG13 fecal swab to the corresponding data from the bat fecal swabs deposited by the same working group using the same methods indicated that the RaTG13 raw data seemed to be different in various aspects. The fecal swab sample showed abnormally less read of bacterial reads in the swab was exceptionally low, i.e. 0.7%, compared to the 20-90% abundance in other fecal swabs from bats processed by similar methods. Also, another raw data in the form of amplicon sequences was deposited in May 2020; however, the dates mentioned on the files of the sequenced amplicons were older (2017, 2018). The genome assembly of RaTG13 could not be done de-novo and the average coverage of the genome ~8%. Also, literature indicates that RaTG13 RBD cannot bind to Rhinolophus ACE-2 receptors. Collectively, the anomalies in the raw data of RaTG13 and other issues pose an important question about the overall authenticity of the RaTG13 genome sequence.

In this letter we investigate if SARS-CoV-2 RNA is involved in the increased ageing of alveolar cells. Our in silico study is explorative. With the results we are able to outline experiments with AEC2 repair of bleomycin damaged alveolar cells. If AEC2 repair capability is diminished by spike RNA then perhaps this result provides a first step on a route to treat immortal lung cancer cells.

SARS-CoV-2 is a novel betacoronavirus and the aetiological agent of the current COVID-19 outbreak that originated in Hubei Province, China. While polymerase chain reaction is the front-line tool for SARS-CoV-2 surveillance, application of amplification-free and culture-free methods for isolation of SARS-CoV-2 RNA, partnered with next-generation sequencing, would provide a useful tool for both surveillance and research of SARS-CoV-2. We here release into the public domain a set of bait capture hybridization probe sequences for enrichment of SARS-CoV-2 RNA from complex biological samples. These probe sequences have been designed using rigorous bioinformatics methods to provide sensitivity, accuracy, and minimal off-target hybridization. Probe design was based on existing, validated approaches for detecting antimicrobial resistance genes in complex samples and it is our hope that this SARS-CoV-2 bait capture platform, once validated by those with samples in hand, will be of aid in combating the current outbreak.

This study presents a methodology to reveal traces of viral particles, as aerosol with known chemical and molecular structure, in a sample by means of photon and electron interactions. The method is based on Monte Carlo simulations and on the analysis of photon-electron fluxes-spectra through energy channels counts as a function of different aerosol viral concentrations in the air sample and looking at the peculiar photon/electron interactions with the potential abnormal atomic hydrogen (H), oxygen (O), carbon (C), and phosphorus (P) compositions present in the air sample as a function of living and nonliving matter with PO4 group RNA/DNA strands in a cluster configuration.

Both SARS-CoV-2 and SARS-CoV initially appeared in China and spread to other parts of the world. SARS-CoV-2 has generated a COVID-19 pandemic causing more than 6 million human deaths worldwide while the SARS outbreak quickly ended in six months with a global total of 774 reported deaths. One of the factors contributing to this stunning difference in the outcome between these two outbreaks is the inaccuracy of the RT-qPCR tests for SARS-CoV-2, which generated a large number of false-negative and false-positive test results that have misled patient management and public health policy-makers. This article presented Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV can in fact detect SARS-CoV-2 accurately due to the well-known nonspecific PCR amplification of DNAs with similar sequences. Using nested RT-PCR followed by Sanger sequencing to retest 50 patient samples collected in January, 2022 and sold as RT-qPCR positive reference confirmed 21 (42%) were false-positive. Although the other 29 positive isolates were categorized as Omicron variant by partial sequencing of the N gene, and the RBD and the NTD of the S gene, 9 (31%) showed focal to complete sequencing failure in the S gene segments due to multi-allelic SNPs. During the course of the study, an Omicron variant isolate containing a BA.1 NTD and a BA.2 RBD in its S gene was also detected. Routine partial S gene sequencing of all PCR-positive samples can timely discover multi-allelic SNPs and viral recombination in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.

Precision medicine seeks to use genomic data to help provide the right treatment to the right patient at the right time. Next-generation sequencing technology allows for the rapid and accurate sequencing of many genes at once. This technology is becoming more common in oncology, though the clinical benefit of incorporating it into precision medicine strategies remains under significant debate. In this manuscript, we discuss the early findings of the impact of next-generation sequencing on cancer patient outcomes. We investigate why not all patients with genomic variants linked to a specific therapy receive that therapy and describe current barriers. Finally, we explore the current state of health insurance coverage for individual genome sequencing and targeted therapies for cancer. Based on our analysis, we recommend increased transparency around the determination of “actionable mutations” and a heightened focus on investigating the variations in health insurance coverage across patients receiving sequencing-matched therapies.

Fast neutron (FN) radiation mediated mutagenesis is a unique approach among the several induced mutagenesis methods being used in plant science in terms of impacted mutations. The FN mutagenesis usually creates deletions from a few bases to several million bases (Mb). A library of random deletion generated using FN mutagenesis lines can provide indispensable resources for the reverse genetic approaches. In this review, information from several efforts made using FN mutagenesis has been compiled to understand the type of induced mutations, frequency, and genetic stability. Concerns regarding the utilization of FN mutagenesis technique for a plant with different level of ploidy and genome complexity are discussed. We have highlighted the utility of next-generation sequencing techniques which can be efficiently utilized for the characterization of mutant lines as well as for the mapping of causal mutations. Pros and cons of Mut-map, MutChromSeq, exon capture, whole genome sequencing, MutRen-Seq, and different tilling approaches can be used for the detection of FN-induced mutation has also been discussed. Genomic resources developed using the FN mutagenesis have been catalogued wooing to meaningful utilization of the available resources. The information provided here will be helpful for the efficient exploration for the crop improvement programs and for better understanding of genetic regulations.

Currently there is little doubt left on the symbiogenetic nature of eukaryotes - genomes of archaeon and bacterium participated in shaping a genome of last eukaryotic common ancestor in equal albeit asymmetric manner, while a merger event itself indicated the advent of a new domain of life. The “symbiogenetic” framework of interaction of two partners is proposed, outlining similar steps essential for three major advents: the origin of life, the origin of complex life and the origin of humans. Given the immense importance of proper energy source for the evolution of life it seems plausible that for any principal increase in complexity, a partnership with a novel energy donor is required. Moreover, a “language” elaborated in the course of communication of partners might have been a prerequisite for a subsequent increase in complexity. Transitions, which led to RNA-protein world, eukaryotes and human brain, resulted in advent of complex languages via communication onsets between two entities in close partnership. Accordingly this further facilitated formation of first cells, multicellular organisms and human society.

Solanesol is a noncyclic terpene alcohol composed of nine isoprene units and it mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). Here, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase, two 1-deoxy-d-xylulose 5-phosphate reductoisomerase, two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase, four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase, two 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, six isopentenyl diphosphate isomerase, and two solanesyl diphosphate synthase (SPS) genes were identified to be involved in solanesol biosynthesis. Furthermore, the two N. tabacum SPS (NtSPS1 and NtSPS2), which had two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs, and leaves from four growing stages, corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthetic genes and the accumulation of solanesol in tobacco.

Green manure could improve soil nutrients and crop production, playing a significant role in sustainable agriculture. However, the impacts of green manure on crop health and the roles soil microbial communities play in the process haven’t been clarified clearly yet. In this study, we investigated soil microbial community composition and structure in four tobacco farmlands, which were treated with different green manure (control, ryegrass, pea and rape), using 16S rRNA gene amplicons sequencing. Results showed that green manure had significant impacts on soil properties, microbial communities and tobacco health. First, soil total C, N and Ca content increased significantly in groups treated with green manure than control. Second, soil community diversity was significantly higher in groups treated with green manure. Third, green manure especially ryegrass, decreased tobacco disease (bacterial wilt) rate dramatically, and the process might be mediated by soil microbial communities. On the one hand, several microbial populations were found to be potentially disease inducible or suppressive. For example, the abundances of Dokdonella and Rhodanobacter were positively correlated to tobacco disease rate, while Acidobacteira_Gp4 and Gp6 had negative correlations with tobacco disease. On the other hand, soil microbial communities were shaped by soil properties (e.g., pH, C and N content). In conclusion, our research showed that green manure could increase soil nutrients directly, and further improve tobacco health mediated by soil microorganisms, which may shed light on revealing interactions among soil properties, microorganisms and plants.

Here we describe ARSA-16S, a tool and accompanying reference database for the analysis of bacterial 16S amplicons. Among other features, ARSA-16S is based on a new model, approach, and algorithm for sequence-level assignment of reads understood as probability distributions, assigns reads individually, and is designed with non-overlapping amplicons covering two non-contiguous regions. A new set of primers for the amplification and sequencing of the V4 and V6 regions is also provided.

Neurofibromatosis type1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1gene. Although congenital pseudarthrosis of the tibia (CPT) has frequently been associated with NF1, the underlying molecular mechanism of CPT in these NF1 patients is yet ill-understood. The aim of the present study was to detect NF1 mutations from genomic DNA and to harbor variants associated with CPT in NF1 patients. Whole-exome sequencing was first carried out with samples from two patients with CPT in one NF1 family, and a novel mutation c.2324A>G (p.E775G) in NF1 gene was identified. Additionally, a missense variant c.455C>T (p.P152L) in BCOR gene completely co-segregated with the CPT phenotype within this family. Subsequently, NF1 and NF2 genes in four other unrelated patients with both NF1 and CPT were screened using targeted sequencing. Four mutations in NF1 gene, including two known mutations (c.2288T>C/p.L763P, c.574 C>T/p.R192*) and two novel mutations (c.768delT/p.F256Lfs*25, c.2229_2230delTG/ p.V744Qfs*23) were detected. Further study confirmed that CPT was present in NF1 families, and NF1 mutations were closely associated with these complex phenotypes. Moreover, the data from the current study indicated that male gender might be a susceptibility factor for CPT in NF1. Therefore, we speculated that BCOR variants might be related to CPT phenotype among male NF1 patients.

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.

All life on Earth uses three integrated molecular systems in which genetic information contained in DNA base sequences is transmitted to ribosomes by RNA and a genetic code, then translated into the amino acid sequences of structural and catalytic proteins. Therefore, the most important point for understanding the origin of life is to determine how such systems could emerge from random processes on the early Earth. In this review, two alternatives are compared: the RNA world hypothesis and the [GADV]-protein world hypothesis. [GADV] refers to four amino acids, Gly [G], Ala [A], Asp [D] and Val [V] that are conserved in the amino acid sequences of many common proteins. Here I will argue that the origins of the three primary processes required for life to begin can be better explained by the GADV hypothesis than the RNA world hypothesis. The GADV hypothesis also incorporates a conversion process by which random polymers can evolve into proteins with ordered sequences.

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.

MicroRNAs (miRNAs) are crucial regulatory RNAs, originated from hairpin precursors. For the past decade, researchers are focusing extensively on miRNA profiles in various plants. However studies on precursor miRNAs (pre-miRNAs) global profiling stay static even in model plants. Here, for the first time in a non-model plant, Abelmoschus esculentus with negligible genome information, we are reporting the global profiling to characterize the miRNAs and their associated pre-miRNAs by applying next generation sequencing approach. Preliminarily we performed sRNA (small RNA) sequencing with five biological replicates of leaf samples to attain 207,285,863 reads and data analysis using miRPlant keyed out 128 known and 845 novel miRNA candidates. With the objective to seize their associated hairpin precursors, we accomplished pre-miRNA sequencing to attain 83,269,844 reads. The paired end reads are merged, adaptor trimmed and the resulting 40-241 nt (nucleotide) sequences were picked out for analysis by using perl scripts from miRGrep tool and in-house built shell script for Minimum Fold Energy Index (MFEI) calculation. Applying stringent criteria of dicer cleavage pattern and perfect stem loop structure revealed precursors for 57 known miRNAs of 15 families and 18 novel miRNAs. Quantitative Real Time (qRT) PCR was performed to determine the expression of selected miRNAs.

Plant life-long organogenesis involves sequential, time and tissue specific expression of developmental genes. This requires activities of Polycomb Group (PcG) and trithorax Group complexes, respectively responsible for repressive Histone 3 trimethylation at lysine 27 (H3K27me3) and activation-related H3K4me3. However, the genome-wide dynamics in histone modifications that occur during developmental processes have remained elusive. Here, we report the distributions of H3K27me3 and H3K4me3 along with transcriptional changes, in a developmental series including Arabidopsis leaf and three stages of flower development. We found that chromatin mark levels are highly dynamic over the time series on nearly half of all Arabidopsis genes. Moreover, during early flower morphogenesis, changes in H3K4me3 prime over changes in H3K27me3 and quantitatively correlate with transcription changes, while H3K27me3 changes occur after prolonged expression changes. Notably, early activation of PcG target genes is dominated by increases in H3K4me3 while H3K27me3 remains present at the locus. Our results reveal H3K4me3 as greater predictor over H3K27me3 for transcription dynamics, unveil unexpected chromatin mechanisms at gene activation and underline the relevance of tissue-specific temporal epigenomics.

The SARS (severe acute respiratory syndrome)-CoV (Coronavirus)-2 S(spike)-protein mRNA/cDNA currently being used as vaccines are antigenic but not antigens against SARS-CoV-2, that causes COVID (Coronavirus Disease) -19. Furthermore, the mRNA and cDNA antigenic vaccines also have potentials for homologous as well as heterologous recombination, primarily into the somatic cell DNA of the vaccine recipients. On the contrary, a SARS-CoV-2 RBD-protein antigen, a part of the S-protein, will directly stimulate antibody production against SARS-CoV-2. Hence, a vaccine composed of SARS-CoV-2 RBD-protein as a safer, fast acting, and effective vaccine against SARS-CoV-2 and thus against COVID-19. This is also useful for some immune compromised individuals.

The COVID-19 pandemic affects all parameters, especially health care professionals, drugs and medical supplies. The KERRA is a mixed medicinal plant capsule that is used for the treatment of patients with high fever with food and drug administration approved by FDA Thailand. Recently, KERRA showed quicker recovery for COVID-19 patients. Therefore, it is possible that some ingredients in KERRA could inhibit SARS-CoV-2. In this study, two important replication-related enzymes in SARS-CoV-2, a main protease and an RNA-dependent RNA polymerase (RdRp), were used to study the effect of KERRA. The results showed that KERRA inhibited the SARS-CoV-2 main protease and SARS-CoV-2 RdRp with IC50 values of 49.91 ± 1.75 ng/mL and 36.23 ± 5.23 µg/mL, respectively. KERRA displayed no cytotoxic activity on macrophage cells at concentrations lower than 1 mg/mL and exhibited anti-inflammatory activity. Additionally, KERRA was against a feline coronavirus (feline infectious peritonitis [FIP]) infection with an EC50 value of 134.3 g/mL. This study supports the potential use of KERRA as a candidate drug for COVID-19.

One consequence of the global technological advancement in conventional metallurgy is the fast depletion rate of valuable minerals, which are also becoming increasingly difficult to find in pure and economically viable forms. This has spurred more interest in technologies that investigate ability of different microorganisms to mobilize valuable metals from their ores via diverse metabolic processes. This study was carried out therefore to isolate, identify and characterize iron solubilizing bacteria from Iron stones of Agbaja iron ore mining site of Kogi State, Nigeria. Crushed samples in the range of 0.25µm and 0.75µm particle sizes were cultured in a modified 9k media to facilitate bacterial growth and pure cultures were then isolated and sub-cultured for further bioleaching studies. Morphological and biochemical analysis suggests that some of the bacteria identified are members of Acidithiobacillus spp, Pseudomonas spp, and Leptospirillum spp. Studies conducted on pure cultures and mixed consortium of the identified organisms shows that a mixture of the three organisms leached iron ore to about 96.16%. Also results of growth pattern due to bacteria countafter 24-72hours of incubation ranged between 0.1×10³ cfu/ml and 12.3 ×10³ cfu/ml for Acidithiobacillus spp. The need to explore the molecular characteristics of these organisms with a view to generating more information on the quality/quantity of their DNA for future cloning activities was also investigated in this work. DNA was extracted using zymo fungal/bacterial extraction mini prep kit ^TM (cat #6001) and subsequently subjected to 1% agarose gel electrophoresis. Visible bands were obtained with Alpha Innotech Gel Documentation Machine. DNA amplification was carried out using a pettier based thermo cycler PCR machine and electrophoresed on 1.5% agarose gel. Results of the PCR shows a visible band corresponding to 1.5kbp using this primer 27F (5¹-GAGTTTGATCCTGGCTCAG-3¹) and 1492R (5^1-GGTTACCTTGTTACGACT-3¹). DNA purity check shows two of the bacteria possess very good qualities for sequencing for further molecular analysis.

The pandemic respiratory disease COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan in December 2019 and then spread throughout the world; Italy was the most affected European country. Despite the close pet-human contact, little is known about the predisposition of pets to SARS-CoV-2. Among these, felines are the most susceptible. In this study, a domestic cat with clear symptoms of pneumonia, confirmed by Rx imaging, was found to be infected by SARS-CoV-2 using quantitative RT–qPCR from a nasal swab. This is the first Italian study reporting on the request of the scientific community to focus attention on the possible role of pets as a SARS-CoV-2 reservoir. An important question remains unanswered: did the cat die from SARS-CoV-2 infection?

RNA sequencing (RNA-Seq) is a complicated protocol, both in the laboratory in generation of data and at the computer in analysis of results. Several decisions during RNA-Seq library construction have important implications for analysis, most notably strandedness during complementary DNA (cDNA) library construction. Here we clarify bioinformatic decisions related to strandedness in both alignment of DNA sequencing reads to reference genomes and subsequent determination of transcript abundance.

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding the RNA biology. Currently, various methods are available to investigate these interactions, in particular, RNA pulldown assay. In the present paper, a method based on the HaloTag technology is presented that is called Halo-RPD (HaloTag RNA PullDown). The proposed protocol uses plants with stable fusion protein expression and the MagneBeads magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic A. thaliana plants. The key stages described in the paper are as follows: 1) preparation of the magnetic beads 2) tissue homogenization and collection of control samples 3) precipitation and wash of RNA-protein complexes; 4) evaluation of protein binding efficacy; 5) RNA isolation; 6) analysis of the obtained RNA. Recommendations for better NGS assay designs are provided.

We present a case of Capnocytophaga canimorsus septic shock after a dog bite in an immunocompetent individual, where real-time nanopore metagenomic sequencing characterized the microbial agent within 19 hours, with subsequent confirmation using droplet digital PCR. Oral swabs from the dog demonstrated a nearly-identical C. canimorsus isolate by sequencing.

Background: Studies employing next-generation sequencing (NGS) show that the oral fungal community (mycobiome) is far more complex than hitherto thought. However, the role of the oral mycobiome in health and disease, including oral carcinogenesis, has not been explored. Objective: To characterize the mycobiome associated with oral squamous cell carcinoma (OSCC). Methods: Tissue biopsies [cases: 25 OSCC; controls: 27 intra-oral fibro-epithelial polyp (FEP)] were collected from oral and maxillofacial units in Sri Lanka. Total DNA was extracted and subjected to sequencing of the fungal ITS2 region using Illumina’s 2x300 bp chemistry. High quality, non-chimeric merged reads were classified to species level using a BLASTN-algorithm with UNITE’s named species sequences as reference. Downstream analyses were performed using QIIME and LEfSe. Results: 364 species representing 160 genera and 2 phyla (Ascomycota and Basidiomycota) were identified, with Candida and Malassezia making up 48% and 11% of the average mycobiome, respectively. However, only 5 species and 4 genera were detected in ≥50% of the samples. The species richness and diversity were significantly lower in OSCC. At the genus level, Candida, Hannaella and Gibberella were overrepresented in OSCC while Alternaria and Trametes were more abundant in FEP. Species-wise, C. albicans, C. etchellsii and Hannaella luteola-like species were enriched in OSCC while Malassezia restricta, Aspergillus tamarii, Alternaria alternate, Cladosporium halotolerans, and Hanseniaspora uvarum-like species were the most significantly abundant in FEP. Conclusions: A dysbiotic mycobiome dominated by C. albicans was found in association with OSCC. Whether this dysbiosis plays a role in oral carcinogenesis warrants further investigation.

Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) has claimed nearly 180,000 lives and continues to spread. There are currently no approved medications or vaccines for this new coronavirus. Studies have shown that the positive RNA genome of SARS-CoV-2 contains unique features, including a 12-base sequence inserted between the two subunits of viral receptor protein Spike. This inserted sequence facilitates the cleavage of Spike by the cellular proteases Furin and TMPRSS2, leading to the fusion of virus and host cell membranes. Current studies are mostly focused on the SARS-CoV-2 Spike protein and its interacting cellular proteins ACE2, Furin, and TMPRSS2. RNA structural studies are limited and little is known about the potential impact of the 12-base sequence insert on the secondary structure of SARS-CoV-2 genomic RNA and/or its transcripts. Here, by using local and global RNA secondary structure predictions, we show that the novel 12-base insert of SARS-CoV-2 genome likely induces a major RNA secondary structure change.

Directed evolution methods are becoming increasingly popular, as they are extremely powerful toward developing new biomolecules with altered/novel activities, e.g., proteins with new catalytic functions or substrate specificities, and nucleic acids that recognize an intended target. Especially useful are systems that have incorporated continuous evolution, where the protein to be evolved undergoes continuous mutagenesis to evolve a desired trait with little to no input from the researcher once the system is started. However, continuous evolution methods can be challenging to implement in the lab and daunting for researchers to invest time and resources. Our intent is to provide basic information and helpful suggestions that we have gained from our experience with bacterial phage-assisted continuous evolution (PACE). Specifically, we review factors to consider before adopting PACE for a given evolution scheme, different types of selection circuits that can be utilized with particular focus on the PACE-B1H selection system, what optimization of a PACE selection circuit may look like using directed evolution of ME47 as a case study, and additional techniques that may be incorporated into a PACE experiment. With this information, researchers will be better equipped to determine if PACE is a valid strategy to use to evolve their proteins and how to set up a valid selection circuit.

We investigated and quantified bacterial accumulation in facemasks used by the general population, using 16S rRNA (Sanger Sequencing), culture and biochemical analysis along with Rose Bengal staining. Additionally, a systematic overview of the literature on face mask contamination was undertaken. We found an average bacterial load of 4.24 × 10⁴ CFU recovered/mask, with a maximum load of 2.85 × 10⁵ CFU. This maximum is 310 times higher than the limit value for contamination of ventilation system outlet surfaces specified by the German standard VDI 6022. Biochemical and molecular identification predominantly found Staphylococcus species (80%), including Staphylococcus aureus, along with endospore-forming Bacillus spp. Literature reports also indicate contamination of masks by bacterial and fungal opportunists of the genera Acinetobacter, Aspergillus, Alternaria, Bacillus, Cadosporium, Candida, Escherichia, Enterobacter, Enterococcus, Klebsiella (including K. pneumoniae), Micrococcus, Microsporum, Mucor, Pseudomonas, Staphylococcus and Streptococcus. Bacterial counts increase linearly with wearing duration, and prolonged use may affect the skin and respiratory microbiomes, promoting consequential eye, skin, oral and airway conditions. These aspects underscore the urgent need for further research and a risk-benefit analysis in respect of mask use, particularly given their unproven efficacy in disrupting the transmission of respiratory viruses and their adverse social consequences.

Studies on the possible association between bacteria and oral squamous cell carcinoma (OSCC) remain inconclusive, largely due to methodological variations/limitations. The objective of this study was to characterize the species composition as well as functional attributes of the bacteriome associated with OSCC. DNA obtained from 20 fresh OSCC biopsies (cases) and 20 deep-epithelium swabs (matched control subjects) were sequenced for the V1-V3 region using Illumina’s 2x300 bp chemistry. High quality, non-chimeric merged reads were classified to species level using a prioritized BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum was the most significantly overrepresented species in the tumors followed by Pseudomonas aeruginosa and Campylobacter sp. Oral taxon 44, while Streptococcus mitis, Rothia mucilaginosa and Haemophilus parainfluenzae were the most significantly abundant in the controls. Functionally, genes involved in bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors while those responsible for DNA repair and combination, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ribosome biogenesis and glycolysis/gluconeogenesis were significantly associated with the controls. This is the first epidemiological evidence for association of F. nucleatum and P. aeruginosa with OSCC. Functionally, an “inflammatory bacteriome” is enriched in OSSC.

In recent years, the focus of disease resistance and susceptibility studies in cattle have been on determining patterns in the innate immune response of key proteins, such as Toll-like receptors (TLR). In the bovine genome, there are 10 TLR family members and, of these, TLR2, TLR4 and TLR6 are specialized in recognition of bacterial ligands. Indigenous cattle breeds of Anatolia have been reported to show fewer signs of clinical bacterial infections, such as bovine tuberculosis and mastitis, and it is hypothesized that this might be due to a less stringent genetic selection during breeding. In contrast, Holstein-Friesian cattle have been under strong selection for milk production, which may have resulted in greater susceptibility to diseases. To test this hypothesis, we have compared the TLR2, TLR4 and TLR6 genes of Anatolian Black (AB), East Anatolian Red (EAR), South Anatolian Red (SAR), Turkish Grey (TG), and Holstein (HOL) cattle using Next Generation Sequencing. The SAR breed had the most variations overall, followed by EAR, AB, TG and HOL. TG had the most variations for TLR2 whereas SAR had the most variations in TLR4 and TLR6. We compared these variants with those associated with disease and susceptibility traits. We used exon variants to construct haplotypes, investigated shared haplotypes within breeds and determined candidate haplotypes for disease resistance phenotype in Anatolian cattle breeds.

Phytophthora. infestans is a well known late blight causing oomycetes pathogen. It evolves and adapts to the host background and new fungicide molecules rapidly within a few years of their release, may be due to the predominance of transposable elements in its genome. Frequent and huge applications of fungicides cause environmental concerns. Here we developed target specific RNA interference based molecules, that are capable of effectively reducing the late blight infection. cDNA microarray expression data was used for the selection of genes involved in the early stage of infection process, sporulation etc. The in vitro synthesis of double stranded RNA molecule, targeting SDH, EF-1α, GPI-HAM344, PLD-3 and HSP-90 encoding genes revealed the reduction in growth, sporulation and symptom expression, which were subsequently assessed by culture bioassay, detached leaf assay and topical application methods. The multiple genes targeted dsRNA nano clay sprayed plants showed enhanced disease resistance (4% disease severity) and least sporulation (<1x103), compared to naked dsRNA spray. Use of nano clay was assumed to be involved in the effective delivery, protection and boosting the action of RNAi in potato plants. A significant difference in the growth, sporulation count, disease severity and reduced expression of the genes and confocal microscopy imaging authenticates the effects of SIGS on late blight disease progression. Our research demonstrated that topical dsRNA nano clay spray under the open-air environment could be an alternative to chemical fungicides and transgenic approaches as a novel plant protection strategy for late blight in an environmentally friendly manner.

Presence of mutated genes strongly correlates with incidence of cancer. Decades of research, however, has not yielded any specific causative gene or set of genes for the vast majority of cancers. The Cancer Genome Atlas program was supposed to provide clarity but it only gave much more data without any accompanying insight into how the disease begins and progresses. It may be time to notice that epidemiological studies consistently show that the environment, not genes, has the principal role in causing cancer. Since carcinogenic chemicals in our food, drink, air and water are the primary culprit, we need to look at the biochemistry of cancer, with focus on enzymes which carry out any and all transformations in a cell. In particular, attention should be paid to the rate-limiting enzyme in DNA synthesis, ribonucleotide reductase (RnR) which is tightly linked to tumor growth. Beside the circumstantial evidence that cancer is induced at its vulnerable active-site by various carcinogens, there exists experimental proof of its role in initiating retinoblastoma and HPV-related cervical cancers. Blocking the activity of RnR is a certain way to arrest cancer.

COVID19 is a current pandemic disease due to the novel coronavirus SARS-CoV-2. The scientific community mounted a strong response by accelerating research and innovation, and rapidly setting the basis to the understanding of molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key step of SARS-CoV2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of structural and functional data on key actors of the replicatory machinery of SARS-CoV-2, filling the gaps in the current availability of structural data using homology modelling. Moreover, learning from similar viruses, we collect literature data to reconstruct the pattern of interactions among protein actors of the SARS-CoV-2 RNA polymerase machinery. In this pattern, an important role is played by co-factors, like Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors holding the entire machinery together to enhance the efficiency of RNA replication.

SARS-CoV2 RNA depended RNA polymerase is an essential enzyme for the survival of the virus in hosts as it helps in the replication of viral RNA. There are no human polymerases that share either sequence or structural homology with viral RNA depended RNA polymerase. These make it a good target for inhibitor discovery, as a specific inhibitor cannot cross-react with the human polymerases. We have used virtual screening, docking, binding energy calculation and simulation to show that valproic acid Co-A, a metabolite from prodrug valproic acid, forms stable interaction with nsP12 of CoV. Our results suggest valproic acid Co-A could be a potential inhibitor of nsP12 of SARS-CoV2.

How did life begin on Earth? And is there life elsewhere in the Cosmos? Challenging questions, indeed. The series of conferences established by NoR CEL in 2013, addresses these very same questions. The basis for this paper is the summary report of oral presentations that were delivered by NoR CEL’s network members during the 2018 Athens conference and, as such, disseminates the latest research which they have put forward. More in depth material can be found by consulting the contributors referenced papers. Overall, the outcome of this conspectus on the conference demonstrates a case for the existence of “probable chemistry” during the prebiotic epoch.

Current knowledge of the RNA world indicates two different genetic codes being present throughout the living world. In contrast to non-coding RNAs that are built of repetitive nucleotide syntax, the sequences that serve as templates for proteins share – as main characteristics – a non-repetitive syntax. The differences in their syntax structure is coherent with the difference of the functions they represent. Whereas non-coding RNAs build groups that serve as regulatory tools in nearly all genetic processes, the coding sections represent the evolutionarily successful function of the genetic information storage medium. The DNA genomes themselves are rather inactive, whereas the non-coding RNA domain is highly active, even as non-random genetic innovation operators. This indicates that repetitive syntax is the essential pre-requisite for RNA interactions to install variable RNA-group-identities, whereas the non-repetitive syntax serves as a stable conservation tool for successful selection processes out of RNA-groups cooperation and competition. The interaction opportunities of RNA loops with repetitive syntax are higher than with non-repetitive ones. Interestingly, these two genetic codes resemble the function of all natural languages, i.e., (a) everyday language use for organization and coordination of biotic group behavior, and (b) artificial (instrumental) language use for conservation of blueprints for complex protein-body constructions.

Reconstructing the Tree of Life remains a central goal in biology. Early methods, relying on small numbers of morphological or genetic characters, often yielded conflicting evolutionary histories, undermining confidence in the results. Investigations based on phylogenomics, which use hundreds to thousands of loci for phylogenetic inquiry, have provided a clearer picture of life's history, but certain branches remain problematic. To resolve difficult nodes on the Tree of Life, two recent studies tested the utility of synteny, the conserved collinearity of orthologous genetic loci in two or more organisms, for phylogenetics. Synteny exhibits compelling phylogenomic potential while also raising new challenges. We identify and discuss specific opportunities and challenges that bear on the value of synteny data and other rare genomic changes for phylogenomic studies. Synteny-based analyses of highly contiguous genome assemblies mark a new chapter in the phylogenomic era and the quest to reconstruct the Tree of Life.

Alfalfa, an important legume forage, is an ideal crop for sustainable agriculture and a potential bioenergy plant. Drought, one of the most common environmental stresses, substantially affects plants’ growth, development and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of 4 samples, each with 3 biological replicates. We identified 348 known miRNAs, belonging to 80 miRNA families, from the 12 libraries and 281 novel miRNAs using Mireap software. 18 known miRNAs in roots and 12 known miRNAs in leaves were screened out as drought-responsive miRNAs. Except for miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs were different between roots and leaves in alfalfa. This is the first study discovering miR157a, miR1507, miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 are drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.

In 2015 in Brazil, Zika virus showed features of geographic expansion and potentially increased virulence. In 2016, New York State issued emergency regulations after the World Health Organization declared Zika virus a Public Health Emergency of International Concern. In this study, mosquito surveillance was conducted in Westchester County, New York, to identify Zika virus and other arboviruses. Twenty trap sites were used for surveillance of Aedes albopictus mosquitoes, the Zika virus vector. The Westchester County Department of Health performed testing for Zika, West Nile, Eastern equine encephalitis, and other flaviviruses on 369 batches comprising 8,891 mosquitoes. Aedes albopictus mosquitoes were identified in Nassau, Rockland, Suffolk, Westchester, and New York City. Despite the increased capacity for specimen analysis, Zika virus was not detected. This study provides the first evidence of appropriate Zika virus surveillance. However, the results do not allow determination of the potential mechanism of local vector-to-human transmission of Zika virus among Aedes albopictus mosquitoes. This study adds to the evidence regarding the distribution, emergence, and trapping capabilities of potential Zika virus vectors.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Typically, mouse-adaptation studies are conducted in inbred laboratory strains BALB/c and C57BL/6, which have defects in the antiviral Mx1 gene that results in increased susceptibility to infection and disease severity. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (a.k.a. CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L, and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering these three adaptive PB1 and PA mutations into the parental CA/07 strain, we demonstrated that adaptive mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions, and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

Purpose. Moraxella is an ocular bacterial pathogen isolated in cases of keratitis, conjunctivitis, and endophthalmitis. Gram-negative brick-shaped diplobacilli from ocular specimens, and slow growth in culture, are early indications of Moraxella ocular infection; however, identifying Moraxella to species can be complex and inconsistent. Methods. In this study, bacteria consistent with Moraxella were identified to species using: 1) DNA sequencing coupled with vancomycin susceptibility, 2) MALDI-TOF Mass Spectrometry, and 3) Biolog ID System. Study samples consisted of 9 ATCC Moraxella controls, 82 isolates from keratitis, 21 isolates from conjunctivitis, and 4 isolates from endophthalmitis. Results. The ATCC controls were correctly identified. For keratitis, 66 (80.5%) were identified as M. nonliquefaciens, 7 (9.0%) as M. lacunata, 5 (6%) as M. osloensis, 2 (2.5%) as Acinetobacter lwoffi, 1 (1.0%) as M. bovis/nonliquefaciens, and 1 (1.0%) as M. osloensis/nonliquefaciens. For conjunctivitis, 9 (43.0%) were identified as M. osloensis, 6 (29.0%) as M. nonliquefaciens, 3 (14.3%) as Roseomonas, 2 (9.5%) as Acinetobacter (parvus, junii), and 1 (4.5%) as M. catarrhalis/M. nonliquefaciens. From endophthalmitis, 3 of 4 of the isolates were M. nonliquefaciens. Overall, M. nonliquefaciens and M. osloensis were identified in 70% (75 of 107) and 13% (14 of 107) of cases, respectively, totaling 83% (89 of 107). Conclusions. M. nonliquefaciens and M. osloensis are important bacterial pathogens of the eye as determined by DNA sequencing, MALDI-TOF MS, and Biolog. Although Moraxella catarrhalis is a clinical pathogen, other species of Moraxella appear to have a prominent role in eye infections.

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.

With a large number of annotated non-coding RNAs (ncRNAs), repetitive sequences are found to constitute functional components (termed as repetitive elements) in ncRNAs that perform specific biological functions. Bioinformatics analysis is a powerful tool for improving our understanding of the role of repetitive elements in ncRNAs. This chapter summarizes recent findings that reveal the role of repetitive elements in ncRNAs. Furthermore, relevant bioinformatics approaches are systematically reviewed, which promises to provide valuable resources for studying the functional impact of repetitive elements on ncRNAs.

The origin of life has not been solved as yet, in spit of the time passage more than thirty years from publication of RNA world hypothesis by W. Gilbert (1986), which is based on the “gene/replicator--first” theory. On the contrary, I have proposed [GADV]-protein world hypothesis (GADV hypothesis), assuming that life emerged from [GADV]-protein world, which is grounded on the “protein/metabolism-first” theory. However, two weak points of protein world hypothesis, (i) protein cannot be produced without gene, and (ii) protein cannot be self-replicated, have been frequently pointed out by supporters of RNA world hypothesis. Then, I examined whether the two weak points could be overcome by GADV hypothesis or not. From the results, it was confirmed that (i) [GADV]-protein could be pseudo-replicated in the absence of gene owing to protein 0^th-order structure or [GADV]-amino acids, and (ii) the replication ability is not always required from the beginning but it is sufficient to acquire it at some time point until the emergence of life. Thus, it was concluded that life emerged as [GADV]-protein world hypothesis, which is grounded on the “protein/metabolism-first” theory, expects.

A double-stranded RNA (dsRNA) virus was isolated and characterized from a strain EW220 of the phytopathogenic fungus Botryosphaeria dothidea. The full-length cDNAs of dsRNAs were 6,434 bp and 5,986 bp in size, respectively. The largest dsRNA of BdBRV1 encodes a cap-pol fusion protein that contains part coat protein gene and a RNA-dependent RNA polymerase (RdRp) domain, and the second dsRNA encodes a hypothetical protein. The dsRNA virus was named Botryosphaeria dothidea botybirnavirus 1 (BdBRV1). BdBRV1 contains spherical virions that are 37 nm in diameter consisting of two segments. The structural proteins of BdBRV1 virus particles were 110 kDa, 90 kDa, and 80 kDa, which encoded by dsRNA1 and 2-ORFs. The analysis of genome sequences revealed that the sequences of BdBRV1 shared 99% identity with Bipolaris maydis botybirnavirus 1(BmBRV1). Phylogenetic reconstruction indicated that BdBRV1 and BmBRV1 are phylogenetically related to the genus Botybirnavirus. Importantly, BdBRV1 influences the growth of B. dothidea and confers hypovirulence to the fungal host. BdBRV1 and BmBRV1 were two strains of the same virus. To our knowledge, this is the first report of a botybirnavirus in B. dothidea and our result might be the first funding in the different fungi infected by the same virus.

Emerging paradigms in interferon (IFN) biology suggest a dynamic INF induced interactome that extends through broader Interferon Stimulated Gene (ISG)- induction, which implicates interferon- ISG coordinated cross-talk with mRNA processing, post-translational modification and metabolic processes that underlie pathological (viral, autoimmune and tumor biology) and physiological (stem cell regenerative pathways) processes. INF immune responses can also be triggered by endogenous host-derived molecules that are generated in response to cellular stress or hemostasis imbalance to establish tissue repair and regeneration in first place, however, overactivation or lack of countermeasures can result in host tissue damage. The proteases are integral to viral and tumor pathology, and importantly serine proteases TMPRSS2 and trypsin have been identified as important molecular determinants underlying COVID-19 pathology, and emergence of coronaviruses cultured in vitro, respectively. We propose that pathogen associated proteases can act as novel stress-inducers to facilitate viral- competent immunomodulation. We term it as Protease Induced Transcriptomic/ epi-Transcriptomic Reshaping (PITTR) of host cells to counter cellular stress. We present a novel experimental model and our preliminary findings of trypsin- primed Caco-2 cells (CPT) that result in translational halt comparable to cells grown under serum-starvation conditions (CSS). CPT at escalating trypsin concentration (CPT- EC) induce upregulation of selective proteins that majorly map to ribosomal, RNA transport, and spliceosome ribonucleoproteins (RNPs). The inclusion of proinflammatory IL1-b to CPT (CPT- IL) resulted in global overexpression of proteins comparable to Caco-2 cells cultured in growth-factor rich serum conditions (CFBS), indicating a likely de-repression of trypsin- induced translational halt. Caco-2 cells display abortive interferon proteome under differential trypsin conditions (CPT, CPT-EC and CPT-IL), which is marked by complete lack of INF generation despite induction of intermediate ISGs, suggestive of protease (trypsin)- dependent regulation of INF response. Viruses regulate the proteome of stress granules (SGs) that are induced to cope transient translational halt as a central adaptive response to pathogen induced cellular stress. The integral components of SGs include non-translating mRNA, ribonucleoproteins (RNPs) and RNA binding proteins (RBPs), which together form biological condensates through a biophysical process involving weak electrostatic interactions through intrinsically disordered regions in RBPs resulting in liquid- liquid phase separation. We compared the CPT- EC proteome to the Mammalian Stress Granules Proteome (MSGP) database to explore potential RBPs that could possibly regulate INF response (and could act as potential anti-viral targets). Notably, differentially upregulated RNPs and potential RBPs from ISG family including ADAR and PRKRA, and RNA helicases implicated in viral pathogenesis were found to be upregulated in the CPT- EC proteome further strengthening the role of proteases (trypsin) in regulating INF pathways independent of the pathogen. We propose that the supplementation of viable SARS-CoV-2 viral loads to trypsin- primed host cells could recapitulate an infectious disease model, which may closely phenocopy pathogen- driven inflammation and signaling events. Based on the global downregulation of seven SERPINS (serine protease inhibitors) linked to thromboinflammation in our LCMS profiling data, we support the candidature of serine protease inhibitors for protease mediated viral pathologies. COVID-19 is increasingly linked to coagulopathy and resemblance to Neutrophil Extracellular Trap (NET) related thromboinflammatory features; SERPIN A1AT (alpha 1 antitrypsin) being a potent neutrophil- elastase inhibitor and a negative regulator of coagulation complement pathway may be a promising candidate for establishing hemostasis rebalancing in COVID-19 pathology.

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.

Removal of the proliferation component of gene expression by PCNA adjustment has been addressed in numerous survival prediction studies for breast cancer and all cancers in the TCGA. These studies indicate that widespread co-regulation of proliferation upwardly biases survival prediction when gene selection is performed on a genome-wide basis. In addition, removal of the correlative effects of proliferation does not reduce the random bias associated with survival prediction using random gene selection. Since most cancers become addicted to DNA repair as a result of forced cellular replication, increased oxidation, and repair deficiencies from oncogenic loss or genetic polymorphisms, we pursued an investigation to remove the proliferation component of expression in DNA repair genes to determine survival prediction. This translational hypothesis-driven focus on DNA repair genes is directly amenable to finding new sets of DNA repair genes that could potentially be studied for inhibition therapy. Overall survival (OS) prediction was evaluated in 18 cancers by using normalized RNA-Seq data for 126 DNA repair genes with expression available in TCGA. Transformations for normality and adjustments for age at diagnosis, stage, and PCNA metagene expression were performed for all DNA repair genes. We also analyzed genomic event rates (GER) for somatic mutations, deletions, and amplification in driver genes and DNA repair genes. After performing empirical p-value testing with use of randomly selected gene sets, it was observed that OS could be predicted significantly by sets of DNA repair genes for 61% (11/18) of the cancers. Interestingly, PARP1 was not a significant predictor of survival for any of the 11 cancers. Results from cluster analysis of GERs indicates that the most opportunistic cancers for inhibition therapy may be AML, colorectal, and renal papillary, because of potentially less confounding due to lower GERs for mutations, deletions, and amplifications in DNA repair genes. However, the most opportunistic cancer for inhibition therapy is likely to be AML, since it showed the lowest GERs for mutations, deletions, and amplifications in DNA repair genes. In conclusion, our hypothesis-driven focus to target DNA repair gene expression adjusted for the PCNA metagene as a means of predicting OS in various cancers resulted in statistically significant sets of genes.

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein along with certain mutations in other regions within the S protein as specific variants in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.

Rhizosphere microbiomes which have been implicated to enhance plant growth and yield are modulated or influenced by a couple of environmental factors such as soil type, plant cultivar, climate change and anthropogenic activities. In particular, anthropogenic activity such as the use of nitrogen-based chemical fertilizers is associated with environmental destruction and this call for a more ecofriendly strategy to increase nitrogen level of agricultural land. This feat is attainable by harnessing nitrogen-fixing endophytic and free-living rhizobacteria. Rhizobium, Pseudomonas, Azospirillum and Bacillus have been found to have positive impacts on crops by enhancing both above and belowground biomass and could therefore play positive roles in achieving sustainable agriculture. Thus, it is needful to study these rhizosphere microbiomes with more sophisticated culture-independent technologies such as next generation sequencing (NGS) with the prospect of discovering novel bacteria with plant growth promoting traits. This review is therefore aimed at discussing factors that can modulate rhizosphere microbiomes with focus on the contributions of nitrogen fixing bacteria towards sustainable agricultural development and the techniques that can be used for their study.

Bacterial wilt (BW), caused by Ralstonia solanacearum is one of the major biotic factors limiting tomato production in the humid tropics. Pyramiding of resistance genes through marker-assisted selection is an efficient way to develop durable BW resistant cultivars. Tomato line ‘Hawaii 7996’ (H7996) is a stable and robust resistance source against various R. solanacearum strains. Major BW resistance quantitative trait loci (QTLs) Bwr-12 and Bwr-6, and several minor or strain specific QTLs have been coarse-mapped in this line, but none has been fine-mapped and validated. The objective of the current study was to construct a high density genetic map using single-nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing, fine-map Bwr-12 and Bwr-6 and determine the effects of these QTLs using a near isogenic line (NIL) population. A high density genetic map using 1,604 SNP markers with an average distance of 0.82 cM was developed for 188 F9 recombinant inbred lines derived from the cross H7996 × WVa700. A total of seven QTLs associated with BW resistance to race 1-phylotype I or/and race 3-phylotype II strains were located on chromosomes 6 (Bwr-6.1, 6.2, 6.3 and 6.4) and 12 (Bwr-12.1, Bwr-12.2 and Bwr-12.3) with logarithm of odds (LOD) scores of 6.2-15.6 and 6.2-31.1, explaining 14.2-33.4% and 15.9-53.9% of the total phenotypic variation contributed from H7996, respectively. To validate the genetic effects of the two QTL regions, a set of 80 BC3F3 NILs containing different sections of Bwr-6 with or without Bwr-12 was phenotyped for disease severity after challenge with either race 1-phylotype I Pss4 or race 3-phylotype II Pss1632 BW strains over two seasons. Bwr-6.1 specific to Pss4 and Bwr-6.3 specific to Pss1632 were mapped to an interval of 5.0 cM (P < 0.05) between 6_33,444,000_SLM6-47 and 6_33,868,000_SLM6-124 SNP marker, and to 2.7 cM (P < 0.01) between positions 6_35,949,000 _SLM6-107 to 6_36,750,000_SLM6-82 marker, respectively. In addition, the specific effect of Bwr-12 for resistance to Pss4 (LOD score of 5.8-16.1, P < 0.01) was confirmed and markers for this QTL have already been made available previously.

Single cell RNA-seq (scRNA-seq) profiles conceal temporal and spatial tissue developmental information. De novo reconstruction of single cell temporal trajectory has been fairly addressed, but reverse engineering single cell 3D spatial tissue localization is hitherto landmark based, and de novo spatial reconstruction is a compelling computational open problem. Here we show that a new algorithm - named D-CE - for coalescent embedding of single cell transcriptomic networks can address this open problem. We rely merely on the spatial information encoded in the expression patterns of developmental signal transcription factor (DST) genes, and we find that D-CE of cell-cell association DST-transcriptomic networks reliably reconstructs the Geo-seq or single cell samples’ 3D spatial tissue distribution. Comparison to the novoSpaRC and CSOmap (recent and only available de novo 3D spatial reconstruction methods) on 16 datasets and 681 reconstructions, reveals a significantly distinctive superior performance of D-CE.

Rice grassy stunt virus (RGSV) a member of Tenuivirus family, is very potent and destructive which effects rice crop in many countries, particularly China. Non coding RNAs have important functions in development and epigenetic regulation of gene expression in numerous organsisms. There is three type of small non coding RNAs have been found in eukaryotes, which are small interferring RNAs (siRNAs), microRNAs (miRNAs) and piwi interacting RNAs (piRNAs). Small RNAs (sRNAs) origination is from the infecting virus which is known as virus-derived small interfering RNAs (vsiRNAs), has responsibility for RNA silencing in plants. Virus-induced gene silencing (VIGS) is mainly dependent on RNA silencing (RNAi). Interestingly, RNA silencing happens in plants during viral infections. RNAi technique showed significant results in Nephotettix cincticeps. RNAi technique demonstrated the gene silencing of planthopper Nilaparvata lugens. The proteins P5, pcf4, Dnj, psn5, and pn6 act as potential movement proteins and serve as silencing suppressors for RGSV. VsiRNAs originate from dsRNA molecules which require Dicer-like (DCL) proteins, RNA dependent RNA polymerase (RdRP) proteins, and Argonaute (AGO) proteins. RdRP uses ssRNA for perfect RNA amplification process and can also be used for DCL dependent secondary vsiRNA formation. VSRs interfere with the movement of signals during silencing mechanism. Moreover, intercellular movement of viruses is facilitated by virus-encoded movement protein. RNAi is found in many eukaryotes which are related to transcriptional or post-transcriptional regulation by gene suppression. Transcription is bidirectional in ssDNA viruses which are originated from dsRNA molecules. In this review, we highlighted the biology of Rice grassy stunt virus and its insect vector and its silencing suppressors. This work will be helpful for plant virologists to understand the whole biogenesis mechanism for rice viruses especially RGSV.

This review will explore the four major pillars required for design and development of an saRNA vaccine: antigen design, vector design, non-viral delivery systems, and manufacturing (both saRNA and lipid nanoparticles (LNP)). In will report on the major innovations, preclinical and clinical data reported in the last five years and will discuss future prospects.

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.

New challenges arise in the face of global climate change which impact every ecosystem on earth, including aquatic systems. This is evident in observations made in regard to the world’s oceans, which show trends of incremental changes in ocean surface temperatures, sea levels, and ocean acidity. These environmental shifts impact human resources such as fisheries and aquaculture. In addition, according to the World Bank, the increase in human population will also require more food and nutrient production, which include industries such as aquaculture. With this increasing demand in aquaculture and fisheries, we must develop efficient and productive methods to operate these industries. We can use genetic methods, specifically transcriptomic information to better understand the biology of our source of nutrition. With the advent of RNASeq techniques, we can provide a better understanding about growth and development, immune function and stress, and adaptations. The use of population genetics or (genomics) to detect Single Nucleotide Polymorphisms (SNPs) between populations or closely related species can provide greater insight from stock structure to fishery-induced evolution. In addition, candidate loci can be investigated further to better understanding evolutionary processes, which provide clues on physiological adaptations and gene expression patterns that can help elucidate how these organisms respond to their current environment. In addition, the use of transcriptomic analyses such as differential gene expression can be used to determine resilience in various environmental conditions such as pollution, hypoxic/anoxic conditions, fluctuations in salinity, and temperature extremes. There has been an increase in transcriptomic studies for many aquaculture species, which has aimed at improving our understanding of growth, development, and metabolism, providing vital information for fisheries and aquaculture industries to make adjustments to environmental conditions such as oxygen availability, nutrition, and salinity. All of these aspects provide insightful information for advancing our knowledge of aquaculture, fisheries and conservation management.

A dogma is normally considered as a principle or a belief accepted as an indisputable truth by some individuals and/or groups. Theoretically there can be no dogmas in science, but it has been demonstrated that scientific thought operates by conceptual changes. A dogma therefore can be understood as a concept present at the heart of some contemporary research programmes that need to be altered to overcome paradigms. Here we argue that two ideas relating to emergence of the biological system research need to be re-evaluated. First, is the idea that research programmes about the emergence of the biological system are the same as those of the origin of cells. Cells are strikingly important biological entities, hard core concepts for the entire field of biology. The emergence of the biological system happened much earlier than the origin of cells and thus the First Universal Common Ancestor (FUCA) should be viewed as a great-grandfather to the Last Universal Cellular Ancestor (LUCA); i.e. the latter is the first cellular life form. Second, RNA-world theories are the focus of mainstream research programmes for the origin of life stricto sensu. In the RNA-world view, self-replication of nucleic acids is seen as one of the most relevant events in the pre-biotic world. Without denying the relevance of self-replication, we argue here that the most germane event which occurred in the pre-biotic world was the crosstalk between nucleic acids and peptides. When these two macromolecules started to interact, the singularity that aggregated the complexity required to produce life emerged. Thus, comprehension of the early origins of the translation machinery and the assembly of the genetic code is key. Therefore, the relevance of cell theory and self-replication should be re-evaluated as well as the concept of life itself.

In this work we demonstrate the proof-of-concept of real-time discrimination between patches of serine or isoleucine monomers in the primary structure of custom-engineered, macro-dipole-like peptides, at uni-molecular level. We employed single-molecule recordings to examine the ionic current through the α-hemolysin (α-HL) nanopore, when hydrophilic serine or hydrophobic isoleucine residues, flanked by segments of oppositely charged arginine and glutamic amino acids functioning as a voltage-dependent ‘molecular brake’ on the peptide, were driven at controllable rates across the nanopore. The observed differences in the ionic currents blockades through the nanopore, visible at time resolutions corresponding to peptide threading through the α-HL’s constriction region, was explained by a simple model of the volumes of electrolyte excluded by either amino acid species, as groups of three serine or isoleucine monomers transiently occupy the α-HL. To provide insights into the conditions ensuring optimal throughput of peptide readout through the nanopore, we probed the sidedness-dependence of peptide association to and dissociation from the electrically and geometrically asymmetric α-HL.

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.

The development of nucleic acid sequencing technology and the unprecedented availability of metadata has evidenced that 45% of human genome constituted by transposable elements (TEs) is not only transcriptionally active but also physiologically needed. Aberrant regulation of TEs, and of human retroviral endogenous sequences (HERVs) in particular, associates with several neurologic and autoimmune diseases, including the Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) frequently comorbid with fibromyalgia (FM). However, no study has yet addressed whether abnormal expression of these sequences correlates with FM. The work presented here shows, for the first time, that in fact HERVs of the H, K and W types are overexpressed in the cells of the immune system of FM patients with or without comorbid ME/CFS. The patients with increased HERV expression (N=14) presented increased levels of interferon (INF-β and INF-γ) but unchanged levels of TNF-α. In support of our proposal that TE activation is a contributor to FM, we find that the tRNA pools are decreased in comparison to matched healthy participants (N=14). The findings reported here could explain the flu-like symptoms FM patients present with in the absence of concomitant infections. Future work towards identifying specific genomic loci differentially affected in FM and ME/CFS is granted.

The transition from the Peptide/RNA world to the Protein/RNA world in the hydrothermal vent environment was a major event in the history of life. The advent of proteins utterly changed the conditions of emerging life, representing a watershed in its development. During subsequent translation various protein enzymes emerged driving protocells into a more complex and interconnected system. With their astonishing versatility, the protein enzymes catalyzed crucial biochemical reactions within protocells into more complex biomolecules in diverse metabolic pathways, whereas structural proteins provided strength and permeability in the cell membrane. Four major events followed after availability of various kinds of protein molecules during prebiotic synthesis. These are: (1) the modification of the phospholipid membrane into the plasma membrane; (2) the origin of primitive cytoplasm; (3) the beginnings of the virus world; and (4) the advent of DNA. The first innovation mediated by proteins was the improvement of the cell membrane. The phospholipid membrane was initially evolved in a vent environment from the gradual modification of a fatty acid membrane via an intermediate phosphatidate acid by non-enzymatic reactions. The phospholipid is then synthesized from phosphatidate acid by a series of enzymes. To make the phospholipid membrane more permeable, various protein molecules interacted with the cell membrane. Proteins not only stabilized the wall membrane, but also acted as pumps, preventing some molecules from the protocells from crossing the membrane barriers, while permitting other selected molecules and ions to enter and leave the protocell. The second modification led by proteins is the gradual conversion of the interior of the protocell from a water-like medium into a gel-like cytoplasm, which became the storehouse of a wide range of biomolecules including amino acids, proteins, nucleic acids, ribosomes, as well as salt and water. The third innovation utilizing the newly synthesized proteins was the emergence of the ancient virus world. In the milieu of different kinds of mRNAs in the prebiotic soup, jelly-roll capsid genes originated de novo within genomes of nonviral mRNAs by overprinting. These fragile capsid genes were possibly coated by proteins on the mineral substrate for stability and durability, transforming them into ancient viral particles. These protein coats were random and were not encoded by encased genes. Some protocells might have engulfed these viral particles, when the capsid genes utilized the ribosomes of the host to translate into the appropriate capsid proteins. These capsid proteins then coated the viral genes to make new copies of primordial viruses inside the protocell. Since then, viruses became capsid-encoding organisms. These primordial mRNA viruses parasitized RNA-based protocells, manipulating them to make new copies of themselves. This was the beginning of a relentless war between viruses and their protocellular hosts. The next stage in viral evolution was the emergence of a primitive retrovirus (pre-retrovirus) with a new kind of replicative strategy in a sense that it could turn its RNA into DNA using its own reverse transcriptase enzyme. This is the beginning of the Retro world that facilitated the transition from RNA to DNA genomes. The infection of RNA protocells with pre-retroviruses progressively transferred the RNA genome to a viral DNA genome by retro-transcription. The advent of DNA by the pre-retrovirus marks the fourth innovation, when a number of enzymes had already developed and were utilized by pre-retroviruses. With continued infection, DNA viruses slowly transferred not only their core replication enzymes, such as helicase, primase, and DNA polymerase, to RNA protocells, but also to their DNAs as well. Thus, began the DNA world, when DNA replaced RNA as the major genome of the protocells. With the advent of DNA, replication of information was entirely dissociated from its expression. Because DNA is much more stable than mRNA with more storage capacity, it is a superb archive for information systems in the form of base sequences. DNA progressively took over the replicative storage function of mRNA, leaving the latter for protein synthesis. The new protocell with the DNA genome will diversify into large populations of DNA protocells that will outcompete populations of RNA protocells. Genetic information began to flow from DNA to mRNA to protein in a two-step process involving transcription and translation. In the biological stage, DNA replication was central to the binary fission of the first cell, orchestrated by the duplication of genomes and then the division of the parent cell into two identical daughter cells. It was carried out by a set of enzymes that formed a Z-ring at the site of replication. With the onset of binary fission, the population of primitive cells grew rapidly in the hydrothermal vent environment, undergoing Darwinian evolution and diversification. These primordial hyperthermophiles, presumably the first life, obtained food and energy directly from the vent environment. However, such a situation was self-limiting, so the early cells evolved their own mechanisms for generating metabolic energy and synthesizing the molecules necessary for their reproduction. The earliest fossil record (≥ 3.5 Ga) of biotic activity is preserved in the Archean hydrothermal and sedimentary rocks of the Nuvvuagittuq Craton of Canada, the Isua Craton of Greenland, the Pilbara Craton of Australia, the Kaapvaal Craton of South Africa, and the Singhbhum Craton of India in the form of the carbonaceous remains of microbial cells, cellular microfossils, and stromatolites. These microscopic fossils provide crucial evidence of the origin and early evolution of prokaryotic cells, beginning with hyperthermophiles. Molecular phylogenetic analysis suggests that both domains of life ¬– Bacteria and Archaea probably split from the last universal common ancestor (LUCA), a hyperthermophilic organism. In the younger sequences of these Archean cratons, two kinds of photosynthetic bacteria, anoxygenic green sulfur bacteria, and oxygenic cyanobacteria, appeared in quick succession from the thermophilic ancestor, indicating a shift of niche from a benthic to a planktonic, with reduced thermotolerance. The development of anoxygenic and oxygenic photosynthesis would have allowed life to escape the hydrothermal setting and invade a newly evolved habitat—broad continental shelves to tap solar energy. Cyanobacteria invaded the global ocean, turned it into blue and green, produced oxygen for the first time, and left their signatures in the carbonates and stromatolites.

Nuclear export of viral mRNAs, is an essential step in the HIV replication cycle. This role is played by a small regulatory protein of HIV-1 called Rev.The N-terminal region of Rev contains an arginine-rich sequence. The arginine-rich motif (ARM) is located between amino acids 38-50 and forms an alpha-helical secondary structure. Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev Response element (RRE). The major viral proteins are not produced if this transport of RNA is stopped. Therefore, knowledge of Rev structure is essential for understanding of its cooperative binding to the RRE, for understanding the mechanism of HIV infection and for the development of antiviral drugs that interfere with Rev’s essential functions and for acknowledgment of good candidate drugs for treatment of AIDS. To understand how REV interact with RRE element of HIV-RNA and its formation of oligomeric complex it is better to characterize the domain wise structure of REV with regard in function of each domain. Due to lack of structural data on Rev no single compound is reported as inhibitor of REV expect antiviral drugs. Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev Protein is much more important. The ARM is a highly specific sequence which allows for the multimerization of Rev and also binding of REV with RNA. Here we are first time exploring the structural characteristics of REV protein both in free form and in complex with RNA at domain function level especially explore the role of ARM motif in REV HIV-1 protein as RNA binding sites by molecular dynamics (MD) simulation and homology modeling studies. Results indicate that the arginine-rich motif (ARM) is crucial in stability of this complex. The residues ARG38, 39, 41, 43, 44, 48, 50, and ASN40 are most interacting with nucleobases of RRE in Crystal structure of Rev and Rev-response-element RNA complex. Our study plays a major role in elaboration of binding of RNA with REV and pave the way for further investigation for therapeutically agent for HIV.

Tilapia lake virus (TiLV) is the main tilapia-infecting virus worldwide, causing serious economic losses. However, there is no vaccine for this viral disease. Here, TiLV ORF10 (TiLV-ORF10) encoding a protein with abundant epitopes was constructed into the eukaryotic expression vector pcDNA3.1, and used to evaluate the immune protective effects in Nile tilapia (Oreochromis niloticus). RT-PCR and western blot analyses confirmed vaccine plasmid expression in tilapia muscle tissues. Moreover, the transcription levels of immunoglobulin M, toll-like receptor 2, myeloid differentiation factor 88, interleukin 8, tumor necrosis factor alpha, gamma-IFN, and nuclear factor κB immune-related genes were statistically significantly upregulated in the spleen, liver, and kidney of vaccinated tilapias (P < 0.05). TiLV challenge experiments showed that relative percent survival (RPS) was significantly enhanced in fish by this DNA vaccine. Moreover, RPS was enhanced further when using a higher amount of the DNA vaccine (85.72% RPS at a DNA dose of 45 μg pcDNA3.1–ORF10). Vaccination with pcDNA3.1–ORF10 significantly reduced virus replication, as evidenced by the low amount of virus in the spleen, liver, and kidney of vaccinated tilapias after TiLV challenge. Thus, pcDNA3.1–ORF10 could induce protective immunity in tilapia and may be a potential vaccine candidate for controlling diseases caused by TiLV.

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.

The 'big data revolution' has enabled novel types of analyses in the life sciences, facilitated by public sharing and reuse of datasets. Here, we review the prodigious potential of reusing publicly available datasets and the challenges, limitations and risks associated with it. Possible solutions to issues and research integrity considerations are also discussed. Due to the prominence, abundance and wide distribution of sequencing data, we focus on the reuse of publicly available sequence datasets. We define ‘successful reuse’ as the use of previously published data to enable novel scientific findings and use selected examples of such reuse from different disciplines to illustrate the enormous potential of the practice, while acknowledging their respective limitations and risks. A checklist to determine the reuse value and potential of a particular dataset is also provided. The open discussion of data reuse and the establishment of the practice as a norm has the potential to benefit all stakeholders in the life sciences.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the etiological agent of the current pandemic worldwide. The pathological condition induced by this pathogen is known as COVID-19 disease. SARS-CoV-2 associated pandemic has been defined as a “public health emergency of international concern” by the International Health Regulation Emergency Committee of the World Health Organization. To date, considerable efforts are in progress to develop more advanced strategies against SARS-CoV-2. Despite the numerous scientific studies published, our knowledge regarding this pathogen is still incomplete, as this virus has been identified only recently. Therefore, scientific investigation of the SARS-CoV-2 has been possible only for a short period of time and effective management of the serious forms of this disease is still lacking. Considerable efforts are in progress worldwide with the purpose to develop more advanced strategies against this pathogen. In this review, we have analyzed the structural and the biological SARS-CoV-2 characteristics and those of other well-known RNA viruses, with the aim to identify possible similarities and analogies between all these pathogens, may be a very useful approach. These infectious agents have been widely studied since several years ago and, a large series of scientific reports are available in the literature regarding this topic. Therefore, focusing on the Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV) and Influenza viruses (IVs), we have collected their historical data, clinical manifestations, pathogenetic mechanisms and related infections. Taking advantage of the results of our research, we have assembled this narrative review, with the aim to get useful insights and lessons from HIV, HCV and IVs characteristics and, consequently, to transfer the obtained knowledge to the study of SARS-CoV-2 biology. There are well known differences between all these pathogens. In particular, they present a distinct mode of transmission, as SARS-CoV-2 and Influenza viruses are airborne pathogens, whereas HIV and HCV are bloodborne infectious agents. However, these viruses exhibit some potential common clinical manifestations and pathogenetic mechanisms and their understanding may contribute to establishing preventive measures and new therapies against SARS-CoV-2. Accordingly, we have analysed and discussed the following points: 1) the biology, the pathogenesis and the clinical manifestations of SARS-CoV-2, HIV, HCV and IVs in mankind; 2) the onset and spreading of pandemics caused by respiratory viruses according to a perspective historical point of view; 3) the possible development of a persistent SARS-CoV-2 reservoir worldwide; 4) the possibility of SARS-CoV-2 reinfection/reactivation; 5) the possible involvement and impact of climatic factors in increasing the risk of SARS-CoV-2 spreading.

Alphaviruses, flaviviruses, measles viruses and rhabdoviruses are enveloped single-stranded RNA viruses, which have been engineered as expression vector systems for recombinant protein expression and vaccine development. Due to the presence of non-structural genes encoding the replicase complex, a 200,000-fold amplification of viral RNA occurs in the cytoplasm of infected cells providing extreme transgene expression levels, which is why they are named self-replicating RNA viruses. Expression of surface proteins of pathogens causing infectious disease and tumor antigens provide the basis for vaccine development against infectious diseases and cancer. The self-replicating RNA viral vectors can be administered as replicon RNA, recombinant viral particles, or layered DNA/RNA replicons. Self-replicating RNA viral vectors have been applied for vaccine development against influenza virus, HIV, hepatitis B virus, human papilloma virus, Ebola virus and recently coronaviruses, especially SARS-CoV-2 the causative agent of the COVID-19 pandemic. Measles virus and rhabdovirus vector-based SARS-CoV-2 vaccine candidates have been subjected to clinical trials. Moreover, RNA vaccine candidates based on self-amplifying alphaviruses have also been evaluated in clinical settings. Various cancers such as brain, breast, lung, ovarian, prostate cancer and melanoma have also been targeted for vaccine development. Robust immune responses and protection have been demonstrated in animal models. Clinical trials have shown good safety and target-specific immune responses. Ervebo, the VSV-based vaccine against Ebola virus disease has been approved for human use.

After human genome sequencing and rapid changes in genome sequencing methods, we have entered in the era of rapidly accumulating genome-sequencing data. This has poses development of several types of methods for representing results of genome sequencing data. Circular genome visualizations tools are also critical in this area as they provide rapid interpretation and simple visualization of overall data. In the last 15 years, we have seen rapid changes in circular visualization tools after the development of the circos tool with 1–2 tools published per year. Herein we have summarized and revisited all these tools until the third quarter of 2018.

The biosynthesis of DNA inherently competes with RNA synthesis because it depends on the reduction of ribonucleotides (RNA precursors) to 2’-deoxyribonucleotides by ribonucleotide reductase (RNR). Hence, RNA viruses can increase viral RNA production in cells by partially blocking the synthesis of DNA, e.g. by downregulating the mammalian selenoprotein thioredoxin reductase (TR), which normally acts to sustain DNA synthesis by regenerating reduced thioredoxin, a hydrogen donor for RNR. Computational and preliminary experimental evidence supports the hypothesis that a number of pathogenic RNA viruses, including HIV-1, Ebola, Zika, some flu viruses, and SARS-CoV-2, target TR isoforms by antisense. TR knockdown would create a host antioxidant defect that could be partially rectified by increased selenium intake, or be exacerbated by selenium deficiency, contributing to viral pathogenesis. There are several non-selenium-dependent means that viruses might also exploit to slow DNA synthesis, such as targeting RNR itself, or components of the glutaredoxin system, which serves as a backup redox system for RNR. HIV-1 substantially downregulates glutathione synthesis, so it interferes with both the thioredoxin and glutaredoxin systems. Computational results suggest that, like Ebola, SARS-CoV-2 targets TR3 by antisense. TR3 is the only TR isoform that includes an N-terminal glutaredoxin domain, so antisense knockdown of TR3 may also affect both redox systems, favoring RNA synthesis. In contrast, some DNA viruses encode their own glutaredoxins, thioredoxin-like proteins and even RNR homologues – so they are doing just the opposite, favoring DNA synthesis. This is clear evidence that viruses can benefit from shifting the RNA:DNA balance to their advantage.

Post-translational modifications (PTMs) of histone residues shape the landscape of gene expression by modulating the dynamic process of RNAPII transcription. The contribution of particular histone modifications to the definition of distinct RNAPII transcription stages remains poorly characterized in plants. Chromatin Immuno-precipitation combined with next-generation sequencing (ChIP-seq) resolves the genomic distribution of histone modifications. Here, we review histone PTM ChIP-seq data in Arabidopsis thaliana and find support for a Genomic Positioning System (GPS) that guides RNAPII transcription. We review the roles of histone PTM “readers”, “writers” and “erasers”, with a focus on the regulation of gene expression and biological functions in plants. The distinct functions of RNAPII transcription during the plant transcription cycle may in part rely on the characteristic histone PTMs profiles that distinguish transcription stages.

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.

This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by probing wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a circular DNA virus. Key silencing-related genes, specifically dicers, were disrupted in order to dissect the RNA silencing pathway and provide useful information on fungal control. Dicers Dcl-1, Dcl-2, and both Dcl-1/Dcl-2- genes were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, susceptibility to viral infection, and small RNA accumulation compared to the wild-type strain. Disruption of Dcl-1 or Dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double dicer mutant strain exhibited slower growth. To examine the effect of viral infection on strains containing null-mutations of Dcl-1, Dcl-2 or both genes, mutants were transfected with full-length RNA transcripts of a hypovirus SsHV2L and copies of a single-stranded DNA mycovirus- SsHADV-1 as a synthetic virus. Results indicate that the ΔDcl-1/Dcl-2 double mutant which was slow growing without virus infection exhibited much more severe debilitation following virus infection. Altered colony morphology including: reduced pigmentation, significantly slower growth, and delayed sclerotial formation. Additionally, there is an absence of virus-derived small RNAs in the virus-infected ∆Dcl-1/Dcl-2 mutant compared to the virus-infected wild-type strain which displays a high percentage of virus-derived small RNA. The findings of these studies suggest that if both dicers are silenced, invasive nucleic acids which include mycoviruses ubiquitous in nature- can greatly debilitate the virulence of fungal plant pathogens.

Plants grown under fluctuating light impact plant developments compared with those grown under non-fluctuating light conditions. However, our knowledge on the underlying regulatory mechanisms is still quite limited, particularly from the transcriptional perspective. In order to investigate the influence of different light intensity distributions on tomato plant development, we designed three fluctuating light intensity distributions with the non-fluctuating light intensity as control and compared the transcriptional differences after five weeks of treatment. We found plant height and aerial/root weight were significantly reduced under all fluctuating light treatments. Transcriptome analysis revealed that the number of up and down regulated genes had a distinct distribution pattern between different treatments and control. The largest difference between the numbers of down and up regulated genes was found between treatment 1 and 3, reaching to a total of 416 genes. The number and type of the top 20 enriched pathways differed between treatments and control. The largest number of genes enriched was involved in the biosynthesis of secondary metabolites. These results provide insights into the transcriptional regulations of tomato under different light intensity distributions.

The deadliest disease in the world, cancer, kills many people every year. The early detection is the only hope for the survival of malignant cancer patients. As a result, in the preliminary stages of , the diagnosis of cancer biomarkers at the cellular level is critical for improving cancer patient survival rates. For decades, scientists have focused their efforts on the invention of biosensors. Biosensors, in addition to being employed in other practical scenarios, can essentially function as cost effective and highly efficient devices for this purpose. Traditional cancer screening procedures are expensive, time-consuming, and inconvenient for repeat screenings. Biomarker-based cancer diagnosis, on the other hand, is rising as one of the most potential tools for early detection, disease progression monitoring, and eventual cancer treatment. As Biosensor is an analytical device, it allows the selected analyte to bind to the biomolecules being studied (– for example RNA, DNA, tissue, proteins, cells). They can be divided based on the kind of biorecognition or transducer elements on the sensor. Most biosensor analyses necessitate the analyte being labeled with a specific marker. In this review article, the application of distinct variants of biosensors against cancer has been described.

Recently, selection in pigs has been focused on improving the lean meat content in carcasses; this focus has been most evident in breeds constituting a paternal component in breeding. Such sire-breeds are used to improve the meat quantity of cross-breed pig lines. However, even in one breed, a significant variation in the meatiness level can be observed. In the present study, the comprehensive analysis of genes and microRNA expression profiles in porcine muscle tissue was applied to identify the genetic background of meat content. The comparison was performed between whole gene expression and miRNA profiles of muscle tissue collected from two sire-line pig breeds (Piertain, Hampshire). The RNA-seq approach allowed the identification of 627 and 416 differentially expressed genes (DEGs) between pig groups differing in terms of loin weight between Pietrain and Hampshire breeds, respectively. The comparison of miRNA profiles showed differential expression of 57 microRNAs for Hampshire and 34 miRNAs for Pietrain pigs. Next, 43 genes and 18 miRNAs were selected as differentially expressed in both breeds and potentially related to muscle development. According to Gene Ontology analysis, identified DEGs and microRNAs were involved in the regulation of the cell cycle, fatty acid biosynthesis and regulation of the actin cytoskeleton. The most deregulated pathways dependent on muscle mass were the Hippo signalling pathway connected with the TGF-beta signalling pathway and controlling organ size via the regulation of ubiquitin-mediated proteolysis, cell proliferation and apoptosis. The identified target genes were also involved in pathways such as the FoxO signalling pathway, signalling pathways regulating pluripotency of stem cells and the PI3K-Akt signalling pathway. The obtained results indicate molecular mechanisms controlling porcine muscle growth and development. Identified genes (SOX2, SIRT1, KLF4, PAX6 and genes belonging to the transforming growth factor beta superfamily) could be considered candidate genes for determining muscle mass in pigs.

In the post antimicrobial era, increasing attention is paid towards using bacteriophage (phage in short) therapy to control antibiotic-resistant bacteria. The first step in phage therapy applications is isolating highly efficient lytic phages or phage cocktails from various sources. When a double-layer- agar with around 0.7% agar in top agar is employed, it results in a low number of phage isolation with a poor resolution, and in many cases, you miss the phage. To address this problem, a low concentration of agar in top agar is examined for better phage isolation. Here, our results proved the efficiency of isolating phage upon formulating a double-layer agar with 0.3% agar in top agar. A sewage sample was collected then phages were isolated, purified, and spotted on a top layer agar with 0.3% agar. The results showed the possibility of isolating a higher number of phages on 0.3% top agar than 0.7%. The finding advocates using 0.3% top agar for the double-layer agar, as it will provide fast, better, and easy phage screening and isolation.

The vast majority of patients with pancreatic ductal adenocarcinoma (PDAC) suffer cachexia. Although cachexia results from concurrent loss of adipose and muscle tissue, most studies focus on muscle alone. Emerging data demonstrate the prognostic value of fat loss in cachexia. Here we sought to identify the muscle and adipose gene profiles and pathways regulated in cachexia. Matched rectus abdominis muscle and subcutaneous adipose tissue were obtained at surgery from patients with benign conditions (n=11) and patients with PDAC (n=24). Self-reported weight loss and body composition measurements defined cachexia status. Gene profiling was done using Ion proton sequencing. Results were queried against external datasets for validation. 961 DE genes were identified from muscle and 2000 from adipose tissue, demonstrating greater response of adipose than muscle. In addition to known cachexia genes such as FOXO1, novel genes from muscle, including PPP1R8 and AEN correlated with cancer weight loss. All the adipose correlated genes including SCGN and EDR17 are novel for PDAC cachexia. Pathway analysis demonstrated shared pathways but largely non-overlapping genes in both tissues. Age related muscle loss predominantly had a distinct gene profiles compared to cachexia. This analysis of matched, externally validate gene expression points to novel targets in cachexia.

Until the 1950s, developmental dyslexia was defined as a hereditary visual disability, selectively affecting reading without compromising oral or non-verbal reasoning skills. This changed radically after the development of the phonological theory of dyslexia; this not only ruled out any role for visual processing in its aetiology, but also cast doubt on the use of discrepancy between reading and reasoning skills as a criterion for diagnosing it. Here I argue that this theory is set at too high a cognitive level to be explanatory; we need to understand the pathophysiological visual and auditory mechanisms that cause children’s phonological problems. I discuss how the ‘magnocellular theory’ attempts to do this in terms of slowed and error prone temporal processing which leads to dyslexics’ defective visual and auditory sequencing when attempting to read. I attempt to deal with the criticisms of this theory and show how it leads to a number of successful ways of helping dyslexic children to overcome their reading difficulties.

Retinoic acid (RA) is the biologically active metabolite of vitamin A,and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By R-Seq analysis, we identify 482 miRNAs in the regenerating CNS of the mollusc Lymnaea stagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate for the first time that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (Pedal A), whereas expression from the growth cones of another class of respiratory motorneurons (RPA) was absent in vitro. These findings support our hypothesis miRNAs are important regulators of retinoic acid induced neuronal outgrowth and regeneration in regeneration-competent species.

Long noncoding RNA (lncRNA) are implicated in various genetic diseases and cancer, attributed to their critical role in gene regulation. RNA sequencing is used to capture their transcripts from certain cell types or conditions. For some studies, lncRNA interactions with other biomolecules have also been captured, which can give clues to their mechanisms of action. Complementary \textit{in silico} methods have been proposed to predict non-coding nature of transcripts and to analyze available RNA interaction data. Here we provide a critical review of such methods and identify associated challenges. Broadly, these can be categorized as reference-based and reference-free or \textit{ab initio}, with the former category of methods requiring a comprehensive annotated reference. The \textit{ab initio} methods can make use of machine learning classifiers that are trained on features extracted from sequences, making them suitable to predict novel transcripts, especially in non-model species. Machine learning approaches such as Logistic Regression, Support Vector Machines, Random Forest, and Deep Learning are commonly used. Initial approaches relied on basic sequential features to train the model, whereas the use of secondary structural features appears to be a promising approach for functional annotation. However, adding secondary features will result in model complexities, thus demanding an algorithm that can handle it and furthermore, considerably increasing the utilization of computation resources. Computational strategies combining identification and functional annotation which can be easily customized are currently lacking. These can be of immense value to accelerate research in this class of RNAs.

Quantum leaps advances in the single-molecule investigative science have been made possible over the past decades through the implication of nanopores, as versatile components on dedicated biosensors. Here, we employed the nanopore-tweezing technique to capture amino acid-functionalized, peptide-nucleic acids (PNA) with -hemolysin-based nanopores, and correlate the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrate that while the system enables detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability single-molecule sequencing.

Hospital-based programs democratize mRNA therapeutics by facilitating the processes to translate a novel RNA idea from the bench to the clinic. Because mRNA is essentially biological software, therapeutic RNA constructs can be rapidly developed. The generation of small batches of clinical grade mRNA to support IND applications and first-in-man clinical trials, as well as personalized mRNA therapeutics delivered at the point-of-care, is feasible at a modest scale of cGMP manufacturing. Advances in mRNA manufacturing science and innovations in mRNA biology, are increasing the scope of mRNA clinical applications.

Since their discovery in 1993, numerous microRNAs (miRNAs) have been identified in humans and other eukaryotic organisms, and their role as key regulators of gene expression is still being elucidated. It is now known that miRNAs not only play a central role in the processes that ensure normal development and physiology, but they are often dysregulated in various diseases. In this review, we present an overview of the role of miRNAs in normal renal development and physiology, in maladaptive renal repair after injury, and in the pathogenesis of renal parenchymal diseases. In addition, we describe methods used for their detection and their potential as therapeutic targets. Continued research on renal miRNAs will undoubtedly improve our understanding of diseases affecting the kidneys and may also lead to new therapeutic agents.

Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKUI1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to human, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.

High-quality chromosome-scale haplotype sequences— of diploid genomes, polyploid genomes and metagenomes — provide important insights into genetic variation associated with disease and biodiversity. However, whole-genome short read sequencing does not yield haplotype information that spans whole chromosomes directly. Computational assembly of shorter haplotype fragments is required for haplotype reconstruction, which can be challenging owing to limited fragment lengths and high haplotype and repeat variability across genomes. Recent advancements in long-read and chromosome-scale sequencing technologies, alongside computational innovations, are improving the reconstruction of haplotypes at the level of whole chromosomes. Here, we review recent methodological progress in these areas and discuss perspectives that could enable routine high-quality haplotype reconstruction in clinical and evolutionary studies.

B chromosomes (B) are supernumerary elements found in many taxonomic groups. Most B chromosomes are rich in heterochromatin and composed of abundant repetitive sequences, especially transposable elements (TEs). Bs origin is generally linked to the A chromosome complement (A). The first report of a B chromosome in African cichlids was on Astatotilapia latifasciata, which can harbor 0, 1 or 2 B chromosomes. Classical cytogenetics studies found high TE content on the species B chromosome. In this study, we aim to understand TE composition and expression on A. latifasciata genome and its relation to the B chromosome. We use bioinformatics analysis to explore TEs genome organization and also their composition on the B chromosome. Bioinformatics findings were validated by fluorescent in situ hybridization (FISH) and real-time PCR (qPCR). A. latifasciata has a TE content similar to other cichlid fishes and several expanded elements on its B chromosome. With RNA sequencing data (RNA-seq) we showed that all major TE classes are transcribed in brain, muscle and male/female gonads. The evaluation of TE expression between B- and B+ individuals showed that few elements have differential expression among groups and expanded B elements were not highly transcribed. Putative silencing mechanisms may the acting on the B chromosome of A. latifasciata to prevent adverse consequences of repeat transcription and mobilization in the genome.

Moonmilk are cave carbonate deposits that host a rich microbiome including antibiotic-producing Actinobacteria making these speleothems appealing for bioprospecting. Here we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave “Grotte des Collemboles” via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9 to 23% of the total bacterial population. Next to actinobacterial OTUs attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and Pseudonocardia (9 OTUs, ~16%) as the most abundant in terms of absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of OTUs unlinked to the culturable Streptomyces strains previously isolated from the same deposits. 43% of OTUs were shared between the three studied collection points while 34% were exclusive to one deposit indicating that distinct speleothems host their own population despite their nearby localization. This important spatial diversity suggests that prospecting within different moonmilk deposits should result in the isolation of unique and novel Actinobacteria. These speleothems also host a wide range of non-streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for isolating rare Actinobacteria.

The telomerase RNA in yeasts is large, usually >1000 nt, and contains functional elements that have been extensively studied experimentally in several disparate species. Nevertheless, they are very difficult to detect by homology-based methods and so far have escaped annotation in the majority of the genomes of Saccharomycotina. This is a consequence of sequences that evolve rapidly at nucleotide level, are subject to large variations in size, and are highly plastic with respect to their secondary structures. Here we report on a survey that was aimed at closing this gap in RNA annotation. Despite considerable efforts and the combination of a variety of different methods, it was only partially successful. While 27 new telomerase RNAs were identified, we had to restrict our efforts to the subgroup Saccharomycetacea because even this narrow subgroup was diverse enough to require different search models for different phylogenetic subgroups. More distant branches of the Saccharomycotina still remain without annotated telomerase RNA.

For over a century, viruses have left a long trail of evidence implicating them as frequent suspects in the development of type 1 diabetes. Through vigorous interrogation of viral infections in individuals with islet autoimmunity and type 1 diabetes using serological and molecular virus detection methods, and mechanistic studies of virus infected human pancreatic β-cells, the prime suspects have been narrowed down to predominantly human enteroviruses. Here we provide a comprehensive overview of evidence supporting the hypothesised role of enteroviruses in the development of islet autoimmunity and type 1 diabetes. We also discuss concerns over the historical focus and investigation bias toward enteroviruses, and summarise current unbiased efforts aimed at characterising the complete population of viruses (the “virome”) contributing early in life to the development of islet autoimmunity and type 1 diabetes. Finally, we review the range of vaccine and antiviral drug candidates currently being evaluated in clinical trials for the prevention and potential treatment of type 1 diabetes.

In the recent past, there has been a rise in Prostate Cancer (PCa) in Asia, particularly India. Although systematic reviews on PCa have dealt on the genetics, genomics and the environmental influence in causal of PCa, no predictive analytics in comparing the PCa from Caucasian, American to Asian population was attempted. In this review article, we have attempted to elaborate this aspect of PCa and deliberated on challenges related to next generation sequencing methods of PCa’s manifestation when compared to the west.