The Mutational Landscape of SARS-CoV-2.

Mutation research is crucial for detecting and treating SARS-CoV-2 and developing vaccines. Using over 5,300,000 sequences from SARS-CoV-2 genomes and custom Python programs, we analyzed the mutational landscape of SARS-CoV-2. Although almost every nucleotide in the SARS-CoV-2 genome has mutated at some time, the substantial differences in the frequency and regularity of mutations warrant further examination. C>U mutations are the most common. They are found in the largest number of variants, pangolin lineages, and countries, which indicates that they are a driving force behind the evolution of SARS-CoV-2. Not all SARS-CoV-2 genes have mutated in the same way. Fewer non-synonymous single nucleotide variations are found in genes that encode proteins with a critical role in virus replication than in genes with ancillary roles. Some genes, such as spike (S) and nucleocapsid (N), show more non-synonymous mutations than others. Although the prevalence of mutations in the target regions of COVID-19 diagnostic RT-qPCR tests is generally low, in some cases, such as for some primers that bind to the N gene, it is significant. Therefore, ongoing monitoring of SARS-CoV-2 mutations is crucial. The SARS-CoV-2 Mutation Portal provides access to a database of SARS-CoV-2 mutations.

A new profiling approach for DNA sequences based on the nucleotides' physicochemical features for accurate analysis of SARS-CoV-2 genomes.

BACKGROUND: The prevalence of the COVID-19 disease in recent years and its widespread impact on mortality, as well as various aspects of life around the world, has made it important to study this disease and its viral cause. However, very long sequences of this virus increase the processing time, complexity of calculation, and memory consumption required by the available tools to compare and analyze the sequences. RESULTS: We present a new encoding method, named PC-mer, based on the k-mer and physic-chemical properties of nucleotides. This method minimizes the size of encoded data by around 2 k times compared to the classical k-mer based profiling method. Moreover, using PC-mer, we designed two tools: 1) a machine-learning-based classification tool for coronavirus family members with the ability to recive input sequences from the NCBI database, and 2) an alignment-free computational comparison tool for calculating dissimilarity scores between coronaviruses at the genus and species levels. CONCLUSIONS: PC-mer achieves 100% accuracy despite the use of very simple classification algorithms based on Machine Learning. Assuming dynamic programming-based pairwise alignment as the ground truth approach, we achieved a degree of convergence of more than 98% for coronavirus genus-level sequences and 93% for SARS-CoV-2 sequences using PC-mer in the alignment-free classification method. This outperformance of PC-mer suggests that it can serve as a replacement for alignment-based approaches in certain sequence analysis applications that rely on similarity/dissimilarity scores, such as searching sequences, comparing sequences, and certain types of phylogenetic analysis methods that are based on sequence comparison.

Conserved Characteristics of NMPylation Activities of Alpha- and Betacoronavirus NiRAN Domains.

Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.

Electrical Detection Assay Based on Programmable Nucleic Acid Probe for Efficient Single-Nucleotide Polymorphism Identification.

The large-scale pandemic and fast evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have triggered an urgent need for an efficient and sensitive on-site nucleic acid testing method with single-nucleotide polymorphism (SNP) identification capability. Here, we report a multiplexed electrical detection assay based on a paperclip-shaped nucleic acid probe (PNprobe) functionalized field-effect transistor (FET) biosensor for highly sensitive and specific detection and discrimination of SARS-CoV-2 variants. The three-stem structure of the PNprobe significantly amplifies the thermodynamic stability difference between variant RNAs that differ in a single-nucleotide mutation. With the assistance of combinatorial FET detection channels, the assay realizes simultaneously the detection and identification of key mutations of seven SARS-CoV-2 variants, including nucleotide substitutions and deletions at single-nucleotide resolution within 15 min. For 70 simulated throat swab samples, the multiplexed electrical detection assay shows an identification accuracy of 97.1% for the discrimination of SARS-CoV-2 variants. Our designed multiplexed electrical detection assay with SNP identification capability provides an efficient tool to achieve scalable pandemic screening.

Modeling SARS-CoV-2 nucleotide mutations as a stochastic process.

This study analyzes the SARS-CoV-2 genome sequence mutations by modeling its nucleotide mutations as a stochastic process in both the time-series and spatial domain of the gene sequence. In the time-series model, a Markov Chain embedded Poisson random process characterizes the mutation rate matrix, while the spatial gene sequence model delineates the distribution of mutation inter-occurrence distances. Our experiment focuses on five key variants of concern that had become a global concern due to their high transmissibility and virulence. The time-series results reveal distinct asymmetries in mutation rate and propensities among different nucleotides and across different strains, with a mean mutation rate of approximately 2 mutations per month. In particular, our spatial gene sequence results reveal some novel biological insights on the characteristic distribution of mutation inter-occurrence distances, which display a notable pattern similar to other natural diseases. Our findings contribute interesting insights to the underlying biological mechanism of SARS-CoV-2 mutations, bringing us one step closer to improving the accuracy of existing mutation prediction models. This research could also potentially pave the way for future work in adopting similar spatial random process models and advanced spatial pattern recognition algorithms in order to characterize mutations on other different kinds of virus families.

TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication.

Coronaviruses are positive-strand RNA viruses with 3' polyadenylated genomes and subgenomic transcripts. The lengths of the viral poly(A) tails change during infection by mechanisms that remain poorly understood. Here, we use a splint-ligation method to measure the poly(A) tail length and poly(A) terminal uridylation and guanylation of the mouse hepatitis virus (MHV) RNAs. Upon infection of 17-CL1 cells with MHV, a member of the Betacoronavirus genus, we observe two populations of terminally uridylated viral transcripts, one with poly(A) tails ~44 nucleotides long and the other with poly(A) tails shorter than ~22 nucleotides. The mammalian terminal uridylyl-transferase 4 (TUT4) and terminal uridylyl-transferase 7 (TUT7), referred to as TUT4/7, add non-templated uracils to the 3'-end of endogenous transcripts with poly(A) tails shorter than ~30 nucleotides to trigger transcript decay. Here we find that depletion of the host TUT4/7 results in an increased replication capacity of the MHV virus. At late stages of infection, the population of uridylated subgenomic RNAs with tails shorter than ~22 nucleotides is reduced in the absence of TUT4/7 while the viral RNA load increases. Our findings indicate that TUT4/7 uridylation marks the MHV subgenomic RNAs for decay and delays viral replication.

MXenes - Versatile 2D materials for identification of biomarkers and contaminants in large scale environments - A review.

Recent years have seen a lot of interest in transition metal carbides/carbonitrides (MXenes), Which is one of newly proliferating two-dimensional (2D) materials.The advantages and applications of synthesizing MXenes-based biosensing systems are interesting. There is an urgent requirement for synthesis of MXenes. Through foliation, physical adsorption, and interface modification,it has been proposed that many biological disorders are related to genetic mutation. Majority of mutations were discovered to be nucleotide mismatches. Consequently, accurate -nucleotide mismatched discrimination is crucial for both diagnosing and treating diseases. To differentiate between such a sensitivealterations in the DNA duplex, several detection methods, particularly Electrochemical-luminescence (ECL) ones, have really been investigated.Mn+1XnTx is common name for MXenes, a novel family of two-dimensional (2D) transition metal carbides, nitrides, and carbonitrides, where T stands for interface termination units (i.e. = O, OH, and/or F). These electronic characteristics of MXenes may be changed between conductive to semiconducting due to abundant organometallic chemistry.Solid-state ECL sensors predicated on MXene would provide the facile nucleotide detection and convenience for usage with minimal training, mobility and possibly minimal cost.This study emphasizes upcoming requirements and possibilities in this area while describing the accomplishments achieved in the usage and employing of MXenes in the research and development of facile biomarkerdetection and their significance in designing electrochemical sensors. Opportunities are addressed for creating 2D MXene materials sensors and devices with incorporated biomolecule sensing. MXenes Carry out this process sensors, address the advantages of using MXenes and their variants as detecting materials for gathering different types of data, and attempt to clarify the design principles and operation of related MXene-based sensors, such as nucleotide detection, Single nucleotide detectors, Cancer theranostics, Biosensing capabilities, Gliotoxin detection, SARS-COV-2 nucleocapsid detection, electrochemical sensors, visual sensors, and humidity sensors. Finally, we examine the major issues and prospects for MXene-based materials used in various sensing applications.

Analysis of SARS-CoV-2 variants from patient specimens in Nevada from October 2020 to August 2021.

In early 2020, the emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population quickly developed into a global pandemic. SARS-CoV-2 is the etiological agent of coronavirus disease 2019 (COVID-19) which has a broad range of respiratory illnesses. As the virus circulates, it acquires nucleotide changes. These mutations are potentially due to the inherent differences in the selection pressures within the human population compared to the original zoonotic reservoir of SARS-CoV-2 and formerly naïve humans. The acquired mutations will most likely be neutral, but some may have implications for viral transmission, disease severity, and resistance to therapies or vaccines. This is a follow-up study from our early report (Hartley et al. J Genet Genomics. 01202021;48(1):40-51) which detected a rare variant (nsp12, RdRp P323F) circulating within Nevada in mid 2020 at high frequency. The primary goals of the current study were to determine the phylogenetic relationship of the SARS-CoV-2 genomes within Nevada and to determine if there are any unusual variants within Nevada compared to the current database of SARS-CoV-2 sequences. Whole genome sequencing and analysis of SARS-CoV-2 from 425 positively identified nasopharyngeal/nasal swab specimens were performed from October 2020 to August 2021 to determine any variants that could result in potential escape from current therapeutics. Our analysis focused on nucleotide mutations that generated amino acid variations in the viral Spike (S) protein, Receptor binding domain (RBD), and the RNA-dependent RNA-polymerase (RdRp) complex. The data indicate that SARS-CoV-2 sequences from Nevada did not contain any unusual variants that had not been previously reported. Additionally, we did not detect the previously identified the RdRp P323F variant in any of the samples. This suggests that the rare variant we detected before was only able to circulate because of the stay-at-home orders and semi-isolation experience during the early months of the pandemic. IMPORTANCE: SARS-COV-2 continues to circulate in the human population. In this study, SARS-CoV-2 positive nasopharyngeal/nasal swab samples were used for whole genome sequencing to determine the phylogenetic relationship of SARS-CoV-2 sequences within Nevada from October 2020 to August 2021. The resulting data is being added to a continually growing database of SARS-CoV-2 sequences that will be important for understanding the transmission and evolution of the virus as it spreads around the globe.

Single Nucleotide Variants (SNVs) of Angiotensin-Converting Enzymes (ACE1 and ACE2): A Plausible Explanation for the Global Variation in COVID-19 Prevalence.

Background: Although it is common knowledge that the coronavirus disease of 2019 (COVID-19) and other viral infections have an uneven impact globally, the reasons for this are still indistinct. The absence of equivalent capacities worldwide in screening, testing, and reporting of cases is one of the ideas put forward to explain this discrepancy. The molecular developments are noteworthy, particularly the role played by single nucleotide polymorphisms (SNPs) in ACEs (ACE1 and ACE2). The virus can enter the host cell thanks to the transmembrane protein ACE2, which is a homolog of ACE1. Objectives: With a focus on the I/D genotype of ACE1 and the rs2285666 SNV of ACE2, we elucidated the prevalence of SNPs in ACE1 and ACE2 in various geographic locations. We examined the relationship between these SNPs and the global patterns of COVID-19 prevalence. Methods: 66 of the 127 articles obtained using PubMed, Google Scholar, and Google directly conformed to the search terms; geographical distribution of viral infections, the prevalence of COVID-19, ACE1, ACE2, SNPs, and prevalence of the DD genotype, and rs2285666. Results: The DD genotype of ACE1 and the rs2285666 SNV of ACE2 are vital in their gene expression and contribute greatly to viral disease susceptibility, development, and severity. There was generally a high prevalence of the DD genotype in Europe and America, where COVID-19 had a more devastating effect than in Asia and Africa. The prevalence of the SNV rs2285666 varied in the following order: East Asia> South Asia >America>Europe >Africa. However, there were conflicting agreements in the association of rs2285666 with COVID-19 susceptibility and prevalence. Conclusion: The ACE1 DD genotype and COVID-19 prevalence have been positively linked in a number of studies. The ACE2 rs2285666 SNV, however, has yielded no definitive results. To determine the relationship between these SNVs and COVID-19 incidence, more research is required.

IB80-A Novel Infectious Bronchitis Virus Genotype (GVIII).

Since mid-2015, there has been an increasing number of chicken samples that are positive for infectious bronchitis virus (IBV) in a screening PCR but which do not show positive results in any established, variant-specific PCR tests (793B, QX, D1466, Massachusetts, D274, Italy 02, Arkansas, Variant 2, Q1). Partial sequencing of the viral genome of those samples shows great similarities, but nucleotide similarity in the S1 gene is only about 57%-61% when compared to any other known GI-GVII IBV genotype and lineage. With nucleotide identity in the S1 gene of approximately 80%, the closest related strain in the National Center for Biotechnology Information database (as of March 15, 2020) is the North American PA/1220/98 isolate (AY789942) designated as a unique variant by Valastro et al. in 2016. Due to its divergence from other IBV strains, we propose that strain, designated IB80, is the type strain of a novel IBV genotype GVIII. So far, IB80 has been detected in commercial layer and broiler parent flocks, frequently showing severe drops in egg production as well as in broiler flocks in Europe and beyond.

IB80­un nuevo genotipo del virus de la bronquitis infecciosa (GVIII). Desde mediados del 2015, ha habido un número creciente de muestras de pollo que resultan positivas para el virus de la bronquitis infecciosa (IBV) por la detección mediante PCR de escrutinio, pero que no muestran resultados positivos en ninguna prueba de PCR específica para las variantes establecidas (793B, QX, D1466, Massachusetts, D274, Italia 02, Arkansas, variante 2, Q1). La secuenciación parcial del genoma viral de esas muestras muestra grandes similitudes, pero la similitud de nucleótidos en el gene S1 es solo del 57% al 61% en comparación con cualquier otro genotipo y linaje GI-GVII conocidos del virus de bronquitis. Con una identidad de nucleótidos en el gene S1 de aproximadamente el 80 %, la cepa relacionada más cercana en la base de datos del Centro Nacional de Información Biotecnológica (al 15 de marzo de 2020) es el aislamiento norteamericano PA/1220/98 (AY789942) designado como variante única por Valastro et al. en 2016. Debido a su divergencia con otras cepas del virus de bronquitis infecciosa, se propone que la cepa, denominada IB80, es la cepa tipo de un nuevo genotipo GVIII del virus de bronquitis infecciosa. Hasta ahora, se ha detectado IB80 en parvadas de reproductoras de pollos de engorde y ponedoras comerciales, y con frecuencia muestra disminuciones severas en la producción de huevo, así como en parvadas de pollos de engorde en Europa y otras regiones.

SARS-CoV-2 RdRp uses NDPs as a substrate and is able to incorporate NHC into RNA from diphosphate form molnupiravir.

The coronavirus disease 2019 has been ravaging throughout the world for three years and has severely impaired both human health and the economy. The causative agent, severe acute respiratory syndrome coronavirus 2 employs the viral RNA dependent RNA polymerase (RdRp) complex for genome replication and transcription, making RdRp an appealing target for antiviral drug development. Systematic characterization of RdRp will undoubtedly aid in the development of antiviral drugs targeting RdRp. Here, our research reveals that RdRp can recognize and utilize nucleoside diphosphates as a substrate to synthesize RNA with an efficiency of about two thirds of using nucleoside triphosphates as a substrate. Nucleoside diphosphates incorporation is also template-specific and has high fidelity. Moreover, RdRp can incorporate ß-d-N4-hydroxycytidine into RNA while using diphosphate form molnupiravir as a substrate. This incorporation results in genome mutation and virus death. It is also observed that diphosphate form molnupiravir is a better substrate for RdRp than the triphosphate form molnupiravir, presenting a new strategy for drug design.

Analysis of 3.5 million SARS-CoV-2 sequences reveals unique mutational trends with consistent nucleotide and codon frequencies.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses. METHODS: Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence. RESULTS: We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant. CONCLUSIONS: Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.

Genotyping SARS-CoV-2 Variants Using Ratiometric Nucleic Acid Barcode Panels.

Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.

Trinucleotide Substitutions at Two Locations in the SARS-CoV-2 Nucleocapsid (N) Gene.

The genomes of sarbecoviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), incorporate mutations with short sequence exchanges based on unknown processes. Currently, the presence of such short-sequence exchanges among the genomes of different SARS-CoV-2 lineages remains uncertain. In the present study, multiple SARS-CoV-2 genome sequences from different clades or sublineages were collected from an international mass sequence database and compared to identify the presence of short sequence exchanges. Initial screening with multiple sequence alignments identified two locations with trinucleotide substitutions, both in the nucleocapsid (N) gene. The first exchange from 5'-GAT-3' to 5'-CTA-3' at nucleotide positions 28,280-28,282 resulted in a change in the amino acid from aspartic acid (D) to leucine (L), which was predominant in clade GRY (Alpha). The second exchange from 5'-GGG-3' to 5'-AAC-3' at nucleotide positions 28,881-28,883 resulted in an amino acid change from arginine and glycine (RG) to lysine and arginine (KR), which was predominant in GR (Gamma), GRY (Alpha), and GRA (Omicron). Both trinucleotide substitutions occurred before June 2020. The sequence identity rate between these lineages suggests that coincidental succession of single-nucleotide substitutions is unlikely. Basic local alignment search tool sequence search revealed the absence of intermediating mutations based on single-base substitutions or overlapping indels before the emergence of these trinucleotide substitutions. These findings suggest that trinucleotide substitutions could have developed via an en bloc exchange. In summary, trinucleotide substitutions at two locations in the SARS-CoV-2 N gene were identified. This mutation may provide insights into the evolution of SARS-CoV-2.

Genome-wide detection of human variants that disrupt intronic branchpoints.

Pre-messenger RNA splicing is initiated with the recognition of a single-nucleotide intronic branchpoint (BP) within a BP motif by spliceosome elements. Forty-eight rare variants in 43 human genes have been reported to alter splicing and cause disease by disrupting BP. However, until now, no computational approach was available to efficiently detect such variants in massively parallel sequencing data. We established a comprehensive human genome-wide BP database by integrating existing BP data and generating new BP data from RNA sequencing of lariat debranching enzyme DBR1-mutated patients and from machine-learning predictions. We characterized multiple features of BP in major and minor introns and found that BP and BP-2 (two nucleotides upstream of BP) positions exhibit a lower rate of variation in human populations and higher evolutionary conservation than the intronic background, while being comparable to the exonic background. We developed BPHunter as a genome-wide computational approach to systematically and efficiently detect intronic variants that may disrupt BP recognition. BPHunter retrospectively identified 40 of the 48 known pathogenic BP variants, in which we summarized a strategy for prioritizing BP variant candidates. The remaining eight variants all create AG-dinucleotides between the BP and acceptor site, which is the likely reason for missplicing. We demonstrated the practical utility of BPHunter prospectively by using it to identify a novel germline heterozygous BP variant of STAT2 in a patient with critical COVID-19 pneumonia and a novel somatic intronic 59-nucleotide deletion of ITPKB in a lymphoma patient, both of which were validated experimentally. BPHunter is publicly available from https://hgidsoft.rockefeller.edu/BPHunter and https://github.com/casanova-lab/BPHunter.

A Review of RRx-001: A Late-Stage Multi-Indication Inhibitor of NLRP3 Activation and Chronic Inflammation.

Chronic unresolving inflammation is emerging as a key underlying pathological feature of many if not most diseases ranging from autoimmune conditions to cardiometabolic and neurological disorders. Dysregulated immune and inflammasome activation is thought to be the central driver of unresolving inflammation, which in some ways provides a unified theory of disease pathology and progression. Inflammasomes are a group of large cytosolic protein complexes that, in response to infection- or stress-associated stimuli, oligomerize and assemble to generate a platform for driving inflammation. This occurs through proteolytic activation of caspase-1-mediated inflammatory responses, including cleavage and secretion of the proinflammatory cytokines interleukin (IL)-1ß and IL-18, and initiation of pyroptosis, an inflammatory form of cell death. Several inflammasomes have been characterized. The most well-studied is the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome, so named because the NLRP3 protein in the complex, which is primarily present in immune and inflammatory cells following activation by inflammatory stimuli, belongs to the family of nucleotide-binding and oligomerization domain (Nod) receptor proteins. Several NLRP3 inflammasome inhibitors are in development, all with multi-indication activity. This review discusses the current status, known mechanisms of action, and disease-modifying therapeutic potential of RRx-001, a direct NLRP3 inflammasome inhibitor under investigation in several late-stage anticancer clinical trials, including a phase 3 trial for the treatment of third-line and beyond small cell lung cancer (SCLC), an indication with no treatment, in which RRx-001 is combined with reintroduced chemotherapy from the first line, carboplatin/cisplatin and etoposide (ClinicalTrials.gov Identifier: NCT03699956). Studies from multiple independent groups have now confirmed that RRx-001 is safe and well tolerated in humans. Additionally, emerging evidence in preclinical animal models suggests that RRx-001 could be effective in a wide range of diseases where immune and inflammasome activation drives disease pathology.

Inspection on the Mechanism of SARS-CoV-2 Inhibition by Penciclovir: A Molecular Dynamic Study.

In recent years, humanity has had to face a critical pandemic due to SARS-CoV-2. In the rapid search for effective drugs against this RNA-positive virus, the repurposing of already existing nucleotide/nucleoside analogs able to stop RNA replication by inhibiting the RNA-dependent RNA polymerase enzyme has been evaluated. In this process, a valid contribution has been the use of in silico experiments, which allow for a rapid evaluation of the possible effectiveness of the proposed drugs. Here we propose a molecular dynamic study to provide insight into the inhibition mechanism of Penciclovir, a nucleotide analog on the RNA-dependent RNA polymerase enzyme. Besides the presented results, in this article, for the first time, molecular dynamic simulations have been performed considering not only the RNA-dependent RNA polymerase protein, but also its cofactors (fundamental for RNA replication) and double-strand RNA.

Simultaneous detection of omicron and other SARS-CoV-2 variants by multiplex PCR MassARRAY technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 h. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation.

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.

Bacterial drug-resistance and viability phenotyping upon disinfectant exposure revealed by single-nucleotide resolved-allele specific isothermal RNA amplification.

Disinfectant abuse poses a risk of bacterial evolution against stresses, especially during the coronavirus disease 2019 (COVID-19) pandemic. However, bacterial phenotypes, such as drug resistance and viability, are hard to access quickly. Here, we reported an allele specific isothermal RNA amplification (termed AlleRNA) assay, using an isothermal RNA amplification technique, i.e., nucleic acid sequence-based amplification (NASBA), integrated the amplification refractory mutation system (ARMS), involving the use of sequence-specific primers to allow the amplification of the targets with complete complementary sequences. AlleRNA assay enables rapid and simultaneous detection of the single nucleotide polymorphism (SNP) (a detection limit, a LOD of 0.5 % SNP) and the viability (a LOD of 80 CFU) of the quinolone resistant Salmonella enterica. With the use of AlleRNA assay, we found that the quinolone resistant S. enterica exhibited higher survival ability during exposure toquaternary ammonium salt, 75 % ethanol and peracetic acid, which might be attributed to the upregulation of stress response-associated genescompared with the susceptible counterparts. Additionally, the AlleRNA assay indicated the potential risk in a high-frequency occurrence of viable but nonculturable (VBNC) quinolone resistant S. enterica induced by disinfectants due to the depression of ATP biosynthesis. The excessive usage of disinfectants during the COVID-19 pandemic should be carefully evaluated due to the latent threat to ecological and human health.