Viral Gene Research Articles

Adeno-associated viral vector gene therapy: Challenges for the paediatric hepatologist.

Hepatoxicity associated with recombinant adeno-associated virus gene therapy is being increasingly encountered by hepatologists in tertiary and quaternary referral units due to the recent increase of these therapies for neuromuscular and haematological disorders. The challenges in managing the condition stem from a lack of good-quality evidence on the appropriate protocols for immunosuppressants due to lack of representative animal models. There is a need for protocols for diagnosing and treating hepatotoxicity and this possible with further research to understand the problem and its management. The review also highlights the importance of a multidisciplinary team in managing hepatotoxicity and recommends further research to better identify at-risk individuals, define the extent of the problem and assess the long-term effects of liver injury and immunosuppressants.

Virome and metagenomic sequencing reveal the impact of microbial inoculants on suppressions of antibiotic resistome and viruses during co-composting

Co-composting with exogenous microbial inoculant, presents an effective approach for the harmless utilization of livestock manure and agroforestry wastes. However, the impact of inoculant application on the variations of viral and antibiotic resistance genes (ARGs) remains poorly understood, particularly under varying manure quantity (low 10 % vs. high 20 % w/w). Thus, employing virome and metagenomic sequencing, we examined the influence of Streptomyces-Bacillus Inoculants (SBI) on viral communities, phytopathogen, ARGs, mobile genetic elements, and their interrelations. Our results indicate that SBI shifted dominant bacterial species from Phenylobacterium to thermotropic Bordetella, and the quantity of manure mediates the effect of SBI on whole bacterial community. Major ARGs and genetic elements experienced substantial changes with SBI addition. There was a higher ARGs elimination rate in the composts with low (∼76 %) than those with high manure (∼70 %) application. Virus emerged as a critical factor influencing ARG dynamics. We observed a significant variation in virus community, transitioning from Gemycircularvirus- (∼95 %) to Chlamydiamicrovirus-dominance. RDA analysis revealed that Gemycircularvirus was the most influential taxon in shaping ARGs, with its abundance decreased approximately 80 % after composting. Collectively, these findings underscore the role of microbial inoculants in modulating virus communities and ARGs during biowaste co-composting.

VOGDB-Database of Virus Orthologous Groups.

Computational models of homologous protein groups are essential in sequence bioinformatics. Due to the diversity and rapid evolution of viruses, the grouping of protein sequences from virus genomes is particularly challenging. The low sequence similarities of homologous genes in viruses require specific approaches for sequence- and structure-based clustering. Furthermore, the annotation of virus genomes in public databases is not as consistent and up to date as for many cellular genomes. To tackle these problems, we have developed VOGDB, which is a database of virus orthologous groups. VOGDB is a multi-layer database that progressively groups viral genes into groups connected by increasingly remote similarity. The first layer is based on pair-wise sequence similarities, the second layer is based on the sequence profile alignments, and the third layer uses predicted protein structures to find the most remote similarity. VOGDB groups allow for more sensitive homology searches of novel genes and increase the chance of predicting annotations or inferring phylogeny. VOGD B uses all virus genomes from RefSeq and partially reannotates them. VOGDB is updated with every RefSeq release. The unique feature of VOGDB is the inclusion of both prokaryotic and eukaryotic viruses in the same clustering process, which makes it possible to explore old evolutionary relationships of the two groups. VOGDB is freely available at vogdb.org under the CC BY 4.0 license.

Epinephelus coioides Sec3 promotes Singapore grouper iridovirus infection by negatively regulates immune response

Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90 % mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response.

A Viral-Encoded Homologue of IPS-1 Modulates Innate Immune Signaling During KSHV Lytic Replication.

Modulation of innate immunity is critical for virus persistence in a host. In particular, viral-encoded disruption of type I interferon, a major antiviral cytokine induced to fight viral infection, is a key component in the repertoire of viral pathogenicity genes. We have identified a previously undescribed open reading frame within the Kaposi's sarcoma-associated herpesvirus (KSHV) genome that encodes a homologue of the human IPS-1 (also referred to as MAVS) protein that we have termed viral-IPS-1 (v-IPS-1). This protein is expressed during the lytic replication program of KSHV, and expression of v-IPS-1 blocks induction of type I interferon upstream of the TRAF3 signaling node including signaling initiated via both the RLR and TLR3/4 signaling axes. This disruption of signaling coincides with destabilization of the cellular innate signaling adaptors IPS-1 and TRIF along with a concatenate stabilization of the TRAF3 protein. Additionally, expression of v-IPS-1 leads to decreased antiviral responses indicating a blot to type I interferon induction during viral infection. Taken together, v-IPS-1 is the first described viral homologue of IPS-1 and this viral protein leads to reprogramming of innate immunity through modulation of type I interferon signaling during KSHV lytic replication.

Characterization of three African swine fever viruses from different clinical settings revealed a potential attenuation mechanism

African swine fever (ASF) is an acute and fatal hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV) that currently threatens the pig industry worldwide. Since the 2018 ASF outbreak in China, ASFV has evolved and caused diverse clinical manifestations, such as chronic and asymptomatic infections. Therefore, it is important to understand the molecular mechanisms underlying ASFV attenuation in the field. Here, we isolated three ASFVs from one diseased and two asymptomatic pigs by using primary porcine alveolar macrophages (PAMs) from both domestic pigs and Bama minipigs. The three ASFVs exhibited similar phenotypes in cell culture, including cytopathic effects (CPEs), hemadsorptions (HADs), viral protein expressions and growth curves. Genome sequencing revealed that all three ASFVs were genotype II strains. Genomic comparisons suggested that the disruption of the viral genes MGF360 and MGF110, rather than EP402R and EP153R, is likely involved in the potential attenuation of ASFV via the upregulation of innate immune responses.

Deficiency in Ever2 does not increase susceptibility of mice to pathogenesis by the mouse papillomavirus, MmuPV1.

Epidermodysplasia verruciformis (EV) is a rare genetic skin disorder that is characterized by the development of papillomavirus-induced skin lesions that can progress to squamous cell carcinoma (SCC). Certain high-risk, cutaneous β-genus human papillomaviruses (β-HPVs), in particular HPV5 and HPV8, are associated with inducing EV in individuals who have a homozygous mutation in one of three genes tied to this disease: EVER1, EVER2, or CIB1. EVER1 and EVER2 are also known as TMC6 and TMC8, respectively. Little is known about the biochemical activities of EVER gene products or their roles in facilitating EV in conjunction with β-HPV infection. To investigate the potential effect of EVER genes on papillomavirus infection, we pursued in vivo infection studies by infecting Ever2-null mice with mouse papillomavirus (MmuPV1). MmuPV1 shares characteristics with β-HPVs including similar genome organization, shared molecular activities of their early, E6 and E7, oncoproteins, the lack of a viral E5 gene, and the capacity to cause skin lesions that can progress to SCC. MmuPV1 infections were conducted both in the presence and absence of UVB irradiation, which is known to increase the risk of MmuPV1-induced pathogenesis. Infection with MmuPV1 induced skin lesions in both wild-type and Ever2-null mice with and without UVB. Many lesions in both genotypes progressed to malignancy, and the disease severity did not differ between Ever2-null and wild-type mice. However, somewhat surprisingly, lesion growth and viral transcription was decreased, and lesion regression was increased in Ever2-null mice compared with wild-type mice. These studies demonstrate that Ever2-null mice infected with MmuPV1 do not exhibit the same phenotype as human EV patients infected with β-HPVs.IMPORTANCEHumans with homozygous mutations in the EVER2 gene develop epidermodysplasia verruciformis (EV), a disease characterized by predisposition to persistent β-genus human papillomavirus (β-HPV) skin infections, which can progress to skin cancer. To investigate how EVER2 confers protection from papillomaviruses, we infected the skin of homozygous Ever2-null mice with mouse papillomavirus MmuPV1. Like in humans with EV, infected Ever2-null mice developed skin lesions that could progress to cancer. Unlike in humans with EV, lesions in these Ever2-null mice grew more slowly and regressed more frequently than in wild-type mice. MmuPV1 transcription was higher in wild-type mice than in Ever2-null mice, indicating that mouse EVER2 does not confer protection from papillomaviruses. These findings suggest that there are functional differences between MmuPV1 and β-HPVs and/or between mouse and human EVER2.

Metatranscriptomic analysis reveals dissimilarity in viral community activity between an ice-free and ice-covered winter in Lake Erie.

Winter is a relatively under-studied season in freshwater ecology. The paucity of wintertime surveys has led to a lack of knowledge regarding microbial community activity during the winter in Lake Erie, a North American Great Lake. Viruses shape microbial communities and regulate biogeochemical cycles by acting as top-down controls, yet very few efforts have been made to examine active virus populations during the winter in Lake Erie. Furthermore, climate change-driven declines in seasonal ice cover have been shown to influence microbial community structure, but no studies have compared viral community activity between different ice cover conditions. We surveyed surface water metatranscriptomes for viral hallmark genes as a proxy for active virus populations and compared activity metrics between ice-covered and ice-free conditions from two sampled winters. Transcriptionally active viral communities were detected in both winters, spanning diverse phylogenetic clades of putative bacteriophage (Caudoviricetes), giant viruses (Nucleocytoviricota, or NCLDV), and RNA viruses (Orthornavirae). However, viral community activity metrics revealed pronounced differences between the ice-covered and ice-free winters. Viral community composition was distinct between winters and viral hallmark gene richness was reduced in the ice-covered relative to the ice-free conditions. In addition, the observed differences in viral communities correlated with microbial community activity metrics. Overall, these findings contribute to our understanding of the viral populations that are active during the winter in Lake Erie and suggest that viral community activity may be associated with ice cover extent.IMPORTANCEAs seasonal ice cover is projected to become increasingly rare on large temperate lakes, there is a need to understand how microbial communities might respond to changing ice conditions. Although it is widely recognized that viruses impact microbial community structure and function, there is little known regarding wintertime viral activity or the relationship between viral activity and ice cover extent. Our metatranscriptomic analyses indicated that viruses were transcriptionally active in the winter surface waters of Lake Erie. These findings also expanded the known diversity of viral lineages in the Great Lakes. Notably, viral community activity metrics were significantly different between the two sampled winters. The pronounced differences we observed in active viral communities between the ice-covered and ice-free samples merit further research regarding how viral communities will function in future, potentially ice-free, freshwater systems.

MC4R Localizes at Excitatory Postsynaptic and Peri-Postsynaptic Sites of Hypothalamic Neurons in Primary Culture.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites.

Transcriptome analysis of CpGV in midguts of type II resistant codling moth larvae and identification of contaminant infections by SNP mapping of RNA-Seq data.

CpGV is a highly virulent pathogen of codling moth, and it has been developed into one of the most successful commercial baculovirus biocontrol agents for pome fruit production worldwide. The emergence of field resistance in codling moth to commercial CpGV products is a threat toward the sustainable use of CpGV. In recent years, different types of resistance (type I-III) were identified. For type II resistance, very little is known regarding the infection process. By studying the virus gene expression patterns of different CpGV isolates in midguts of type II-resistant codling moth larvae, we found that the type II resistance mechanism is most likely based on intracellular factors rather than a receptor component. By applying SNP mapping of the RNA-Seq data, we further emphasize the importance of identifying the infective agents in in vivo experiments when activation of a covert infection cannot be excluded.

Genome composition-based deep learning predicts oncogenic potential of HPVs.

Human papillomaviruses (HPVs) account for more than 30% of cancer cases, with definite identification of the oncogenic role of viral E6 and E7 genes. However, the identification of high-risk HPV genotypes has largely relied on lagged biological exploration and clinical observation, with types unclassified and oncogenicity unknown for many HPVs. In the present study, we retrieved and cleaned HPV sequence records with high quality and analyzed their genomic compositional traits of dinucleotide (DNT) and DNT representation (DCR) to overview the distribution difference among various types of HPVs. Then, a deep learning model was built to predict the oncogenic potential of all HPVs based on E6 and E7 genes. Our results showed that the main three groups of Alpha, Beta, and Gamma HPVs were clearly separated between/among types in the DCR trait for either E6 or E7 coding sequence (CDS) and were clustered within the same group. Moreover, the DCR data of either E6 or E7 were learnable with a convolutional neural network (CNN) model. Either CNN classifier predicted accurately the oncogenicity label of high and low oncogenic HPVs. In summary, the compositional traits of HPV oncogenicity-related genes E6 and E7 were much different between the high and low oncogenic HPVs, and the compositional trait of the DCR-based deep learning classifier predicted the oncogenic phenotype accurately of HPVs. The trained predictor in this study will facilitate the identification of HPV oncogenicity, particularly for those HPVs without clear genotype or phenotype.

Blood biodistribution and vector shedding of valoctocogene roxaparvovec in people with severe hemophilia A

AbstractFollowing systemically administered adeno-associated virus gene therapy, vector particles are widely distributed, raising concerns about horizontal or germline vector transmission. Characterization of biodistribution and kinetics of vector DNA in body fluids can address these concerns and provide insights into vector behavior in accessible samples. We investigated biodistribution and vector shedding profile of valoctocogene roxaparvovec in men with severe hemophilia A enrolled in the phase 3 GENEr8-1 trial. Participants (n = 134) received a single 6 × 1013 vector genome (vg)/kg infusion and were assessed over 3 years. Vector DNA was measured using 4 different assays. Total vector DNA was evaluated in blood, saliva, stool, semen, and urine by quantitative polymerase chain reaction (qPCR). Encapsidated vector DNA was measured in plasma and semen with immunocapture-based qPCR. Contiguity of vgs and assembly of inverted terminal repeat fusions were measured in whole blood and peripheral blood mononuclear cells (PBMCs) using multicolor digital PCR. Median peak vector DNA levels observed 1 to 8 days after dosing were highest in blood, followed by saliva, semen, stool, and urine. Concentrations declined steadily. Encapsidated vector DNA cleared faster than total vector DNA, achieving clearance by ≤12 weeks in plasma and semen. Predominant vector genome forms transitioned from noncontiguous to full-length over time in whole blood and PBMCs, indicating formation of stable circularized episomes within nucleated cells. The replication-incompetent nature of valoctocogene roxaparvovec, coupled with steady clearance of total and encapsidated vector DNA from shedding matrices, indicates transmission risk is low. This trial was registered at www.ClinicalTrials.gov as #NCT03370913.

An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome

The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p﹤0.05, t-test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.

Increased expression of human endogenous retrovirus K in endomyocardial biopsies from patients with cardiomyopathy – a transcriptomics meta-analysis

Most studied, investigating transcriptional changes in myocardial biopsies focus on human genes. However, the presence and potential consequence of persistent expression of viral genes within the myocardium is unclear. The aim of the study was to analyze viral gene expression in RNAseq data from endomyocardial biopsies. The NCBI Bioproject library was screened for published projects that included bulk RNA sequencing data from endomyocardial biopsies from both healthy and diseased patients with a sample size greater than 20. Diseased patients with hypertrophic, dilated, and ischemic cardiomyopathies were included. A total of 507 patients with 507 samples from 6 bioprojects were included and mapped to the human genome (hg38). Unmappable sequences were extracted and mapped to an artificial ‘super-virus’ genome comprising 12,182 curated viral reference genomes. Subsequently, the sequences were reiteratively permutated and mapped again to account for randomness. In total, sequences from 68 distinct viruses were found, all of which were potentially human pathogenic. No increase in cardiotropic viruses was found in patients with dilated cardiomyopathy. However, the expression levels of the particle forming human endogenous retrovirus K were significantly increased (q < 0.0003, ANOVA). Higher expression levels were associated with increased expression in mitochondrial pathways such as oxidative phosphorylation (p < 0.0001). In Conclusion, expression of human endogenous retrovirus K is significantly increased in patients with dilated cardiomyopathy, which in turn was associated with transcriptional alterations in major cellular pathways.

Deciphering the evolutionary landscape of severe fever with thrombocytopenia syndrome virus across East Asia.

Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a significant public health challenge in East Asia, necessitating a deeper understanding of its evolutionary dynamics to effectively manage its spread and pathogenicity. This study provides a comprehensive analysis of the genetic diversity, recombination patterns, and selection pressures across the SFTSV genome, utilizing an extensive dataset of 2041 sequences from various hosts and regions up to November 2023. Employing maximum likelihood and Bayesian evolutionary analysis by sampling trees (BEAST), we elucidated the phylogenetic relationships among nine distinct SFTSV genotypes (A, B1, B2, B3, B4, C, D, E, and F), revealing intricate patterns of viral evolution and genotype distribution across China, South Korea, and Japan. Furthermore, our analysis identified 34 potential reassortments, underscoring a dynamic genetic interplay among SFTSV strains. Genetic recombination was observed most frequently in the large segment and least in the small segment, with notable recombination hotspots characterized by stem-loop hairpin structures, indicative of a structural propensity for genetic recombination. Additionally, selection pressure analysis on critical viral genes indicated a predominant trend of negative selection, with specific sites within the RNA-dependent RNA polymerase and glycoprotein genes showing positive selection. These sites suggest evolutionary adaptations to host immune responses and environmental pressures. This study sheds light on the intricate evolutionary mechanisms shaping SFTSV, offering insights into its adaptive strategies and potential implications for vaccine development and therapeutic interventions.

Suppression of HSV-1 infection and viral reactivation by CRISPR-Cas9 gene editing in 2D and 3D culture models

Although our understanding of HSV-1 biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR/Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D hiPSC-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 gene. We found that targeting the ICP0 or ICP27 gene with AAV2-CRISPR/Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR/Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR/Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1 associated complications.

Small RNA sequencing analysis reveals regulation of microRNA expression in Madin-Darby canine kidney epithelial cells infected with Canid alphaherpesvirus 1.

Canid alphaherpesvirus 1 (CHV-1) infection can cause spontaneous abortions in pregnant dams, and in young puppies, fatal systemic infections are common. MicroRNAs (miRNAs) affect viral infection by binding to messenger RNAs, and inhibiting expression of host and/or viral genes. We conducted deep sequencing of small RNAs in CHV-1-infected and mock-infected Madin-Darby Canine Kidney (MDCK) epithelial cells, and detected sequences corresponding to 282 cellular miRNAs. Of these, 18 were significantly upregulated at 12h post-infection, most of which were encoded on the X chromosome. We next quantified the mature forms of several of the miRNAs using stem loop RT-qPCR. Our results revealed a discordance between the levels of small RNAs corresponding to canine miRNAs, and levels of the corresponding mature miRNAs, which suggests a block in miRNA biogenesis in infected cells. Nevertheless, we identified several mature miRNAs that exhibited a statistically significant increase upon infection. These included cfa-miR-8908b, a miRNA of unknown function, and cfa-miR-146a, homologs of which target innate immune pathways and are known to play a role in other viral infections. Interestingly, ontology analysis predicted that cfa-miR-8908b targets factors involved in the ubiquitin-like protein conjugation pathway and peroxisome biogenesis among other cellular functions. This is the first study to evaluate changes in miRNA levels upon CHV-1 infection. Based on our findings, we developed a model whereby CHV-1 infection results in changes in levels of a limited number of cellular miRNAs that target elements of the host immune response, which may provide clues regarding novel therapeutic targets.

Viral communities in a pH>10 serpentinite-like environment: insight into diversity and potential roles in modulating the microbiomes by bioactive vitamin B9 synthesis.

This study presents a comprehensive investigation into the diversity, potential functionalities, and virus-microbe interactions in an artificially induced strongly alkaline environment. Functional validation of the detected viral folA genes encoding dihydrofolate reductase substantiated the synthesis of essential cofactors by viruses, which may be ubiquitous, considering the broad distribution of the viral genes associated with folate cycling.

KRASFormer: a fully vision transformer-based framework for predicting KRAS gene mutations in histopathological images of colorectal cancer

Detecting the Kirsten Rat Sarcoma Virus (KRAS) gene mutation is significant for colorectal cancer (CRC) patients. The KRAS gene encodes a protein involved in the epidermal growth factor receptor (EGFR) signaling pathway, and mutations in this gene can negatively impact the use of monoclonal antibodies in anti-EGFR therapy and affect treatment decisions. Currently, commonly used methods like next-generation sequencing (NGS) identify KRAS mutations but are expensive, time-consuming, and may not be suitable for every cancer patient sample. To address these challenges, we have developed KRASFormer, a novel framework that predicts KRAS gene mutations from Haematoxylin and Eosin (H & E) stained WSIs that are widely available for most CRC patients. KRASFormer consists of two stages: the first stage filters out non-tumor regions and selects only tumour cells using a quality screening mechanism, and the second stage predicts the KRAS gene either wildtype’ or mutant’ using a Vision Transformer-based XCiT method. The XCiT employs cross-covariance attention to capture clinically meaningful long-range representations of textural patterns in tumour tissue and KRAS mutant cells. We evaluated the performance of the first stage using an independent CRC-5000 dataset, and the second stage included both The Cancer Genome Atlas colon and rectal cancer (TCGA-CRC-DX) and in-house cohorts. The results of our experiments showed that the XCiT outperformed existing state-of-the-art methods, achieving AUCs for ROC curves of 0.691 and 0.653 on TCGA-CRC-DX and in-house datasets, respectively. Our findings emphasize three key consequences: the potential of using H & E-stained tissue slide images for predicting KRAS gene mutations as a cost-effective and time-efficient means for guiding treatment choice with CRC patients; the increase in performance metrics of a Transformer-based model; and the value of the collaboration between pathologists and data scientists in deriving a morphologically meaningful model.

Structure of the Bacteriophage PhiKZ Non-virion RNA Polymerase Transcribing from its Promoter p119L

Bacteriophage ΦKZ (PhiKZ) is the founding member of a family of giant bacterial viruses. It has potential as a therapeutic as its host, Pseudomonas aeruginosa, kills tens of thousands of people worldwide each year. ΦKZ infection is independent of the host transcriptional apparatus; the virus forms a “nucleus”, producing a proteinaceous barrier around the ΦKZ genome that excludes the host immune systems. It expresses its own non-canonical multi-subunit non-virion RNA polymerase (nvRNAP), which is imported into its “nucleus” to transcribe viral genes. The ΦKZ nvRNAP is formed by four polypeptides representing homologues of the eubacterial β/β′ subunits, and a fifth that is likely to have evolved from an ancestral homologue to σ-factor. We have resolved the structure of the ΦKZ nvRNAP initiating transcription from its cognate promoter, p119L, including previously disordered regions. Our results shed light on the similarities and differences between ΦKZ nvRNAP mechanisms of transcription and those of canonical eubacterial RNAPs and the related non-canonical nvRNAP of bacteriophage AR9.