High Mutation Rate Research Articles

Contrasting PRRSV temporal lineage patterns at the individual farm, production system, and regional levels in Ohio and neighboring states from 2017 to 2021

Porcine reproductive and respiratory virus (PRRSV), one of the most significant viruses in the swine industry, has been challenging to control due to its high mutation and recombination rates and complexity. This retrospective study aimed to describe and compare the distribution of PRRSV lineages obtained at the individual farm, production system, and regional levels. PRRSV-2 (type 2) sequences (n = 482) identified between 2017 – 2021 were provided by a regional state laboratory (Ohio Department of Agriculture, Animal Disease Diagnostic Center (ODA-ADDL)) collected from swine farms in Ohio and neighboring states, including Indiana, Michigan, Pennsylvania, and West Virginia. Additional sequences (n = 138) were provided by one collaborating swine production system. The MUSCLE algorithm on Geneious Prime® was used to align the ORF5 region of PRRSV-2 sequences along with PRRSV live attenuated vaccine strains (n = 6) and lineage anchors (n = 169). Sequenced PRRSV-2 were assigned to the most identical lineage anchors/vaccine strains. Among all sequences (n = 620), 29.8% (185/620) were ≥ 98.0% identity with the vaccine strains, where 93.5% (173/185) and 6.5% (12/185) were identical with the L5 Ingelvac PRRS® MLV and L8 Fostera® PRRS vaccine strains, respectively, and excluded from the analysis. At the regional level across five years, the top five most identified lineages included L1A, L5, L1H, L1C, and L8. Among non-vaccine sequences with production system known, L1A sequences were mostly identified (64.3% – 100.0%) in five systems, followed by L1H (0.0% - 28.6%), L1C (0.0% – 10.5%), L5 (0.0% – 14.4%), L8 (0.0% – 1.3%), and L1F (0.0% – 0.5%). Furthermore, among non-vaccine sequences with the premise identification available (n = 262), the majority of sequences from five individual farms were either classified into L1A or L5. L1A and L5 sequences coexisted in three farms, while samples submitted by one farm contained L1A, L1H, and L5 sequences. Additionally, the lineage classification results of non-vaccine sequences were associated with their restriction fragment length polymorphism (RFLP) patterns (Fisher’s exact test, p < 0.05). Overall, our results show that individual farm and production system-level PRRSV-2 lineage patterns do not necessarily correspond to regional-level patterns, highlighting the influence of individual farms and systems in shaping PRRSV occurrence within those levels, and highlighting the crucial goal of within-farm and system monitoring and early detection for accurate knowledge on PRRSV-2 lineage occurrence and emergence.

Unravelling the link between SARS-CoV-2 mutation frequencies, patient comorbidities, and structural dynamics.

Genomic surveillance is crucial for tracking emergence and spread of novel variants of pathogens, such as SARS-CoV-2, to inform public health interventions and to enforce control measures. However, in some settings especially in low- and middle- income counties, where sequencing platforms are limited, only certain patients get to be selected for sequencing surveillance. Here, we show that patients with multiple comorbidities potentially harbour SARS-CoV-2 with higher mutation rates and thus deserve more attention for genomic surveillance. The relationship between the patient comorbidities, and type of amino acid mutations was assessed. Correlation analysis showed that there was a significant tendency for mutations to occur within the ORF1a region for patients with higher number of comorbidities. Frequency analysis of the amino acid substitution within ORF1a showed that nsp3 P822L of the PLpro protease was one of the highest occurring mutations. Using molecular dynamics, we simulated that the P822L mutation in PLpro represents a system with lower Root Mean Square Deviation (RMSD) fluctuations, and consistent Radius of gyration (Rg), Solvent Accessible Surface Area (SASA) values-indicate a much stabler protein than the wildtype. The outcome of this study will help determine the relationship between the clinical status of a patient and the mutations of the infecting SARS-CoV-2 virus.

COP9 signalosome complex is a prognostic biomarker and corresponds with immune infiltration in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is among the most common deadly tumors but still lacks specific biomarkers for diagnosis, prognosis, and treatment guidance. The COP9 signalosome (COPS) is an essential regulator of the ubiquitin conjugation pathway upregulated in various cancers. We evaluated the contributions of COPS subunits to HCC tumorigenesis and their utility for prognosis. We comprehensively evaluated the tumor expression pattern and tumorigenic functions of COPS subunits using The Cancer Genome Atlas (TCGA), The Human Protein Atlas and immunohistochemistry. Kaplan-Meier, Cox regression, ROC curve, and nomogram analyses were used to assess the predictive values of COPS subunits for clinical outcome. Expression levels of COPS subunits were significantly upregulated in HCC tissues, which predicted shorter overall survival (OS). Further, Cox regression analysis identified COPS5, COPS7B, and COPS9 as independent prognostic biomarkers for OS. High mutation rates were also found in COPS subunits. Functional network analysis indicated that COPS and neighboring genes regulate 'protein neddylation', 'protein deneddylation', and 'protein ubiquitination'. The COPS PPI included strong interactions with p53, CUL1/2/3/4, and JUN. Moreover, the correlations between COPS subunit expression levels and tumor immune cell infiltration rates were examined using TIMER, TISIDB, ssGSEA, and ESTIMATE packages. COPS subunits expression levels were positively correlated with specific tumor immune cell infiltration rates, immunoregulator expression levels, and microsatellite instability in HCC. Finally, knockout of COPS6 and COPS9 in HCC cells reduced while overexpression enhanced proliferation rate and metastasis capacity. Our study revealed that COPS potential biomarker for unfavorable HCC prognosis and indicators of immune infiltration, tumorigenicity, and metastasis.

A novel immunoinformatics approach for developing a poly-epitope vaccine targeting foot and mouth disease virus, exploiting structural VP proteins

Foot and mouth Disease virus (FMDV) belongs to Picornaviridae family and Aphthovirus genus causing Foot and mouth disease (FMD) in cloven-hoofed animals. FMDV, a prevalent virus induces both acute and chronic infections with high mutation rates resulting in seven primary serotypes, making vaccine development indispensable. Due to time and cost effectiveness of the immunoinformatic approach, we designed in-silico polyepitope vaccine (PEV) for the curtailment of FMDV. Structural and immunogenic parts of FMDV (Viral Protein 1 (VP1), Viral Protein 2 (VP2), Viral Protein 3 (VP3), and Viral Protein 4 (VP4)) were used to design the cytotoxic T Lymphocyte (CTL), Helper T Lymphocyte (HTL), and B-cell epitopes, followed by screening for antigenic, non-allergenic, Interferon (IFN) simulator, and non-toxicity, which narrowed down to 7 CTL, 3 HTL, and 12 B-cell epitopes. These selected epitopes were linked using appropriate linkers and Cholera Toxin B (CTB) adjuvant for immunological modulation. The physiochemical analyses followed by the structure prediction demonstrated the stability, hydrophilicity and solubility of the PEV. The interactions and stability between the vaccine, Toll like Receptor 3 (TLR3) and Toll like receptor 7 (TLR7) were revealed by molecular docking and Molecular Mechanics/Poisson Boltzmann Surface Area (MMPBSA) with high stability and compactness verified by MD simulation. In-silico immune simulation demonstrated a strong immunological response. FMDV-PEV (Poly epitope vaccine) will be effectively produced in an E. coli system, as codon optimization and cloning in an expression vector was performed. The effectiveness, safety, and immunogenicity profile of FMDV-PEV may be confirmed by further experimental validations. Communicated by Ramaswamy H. Sarma

In Vitro Microevolution and Co-Selection Assessment of Amoxicillin and Cefotaxime Impact on Escherichia coli Resistance Development.

The global spread of antimicrobial resistance has become a prominent issue in both veterinary and public health in the 21st century. The extensive use of amoxicillin, a beta-lactam antibiotic, and consequent resistance development are particularly alarming in food-producing animals, with a focus on the swine and poultry sectors. Another beta-lactam, cefotaxime, is widely utilized in human medicine, where the escalating resistance to third- and fourth-generation cephalosporins is a major concern. The aim of this study was to simulate the development of phenotypic and genotypic resistance to beta-lactam antibiotics, focusing on amoxicillin and cefotaxime. The investigation of the minimal inhibitory concentrations (MIC) of antibiotics was performed at 1×, 10×, 100×, and 1000× concentrations using the modified microbial evolution and growth arena (MEGA-plate) method. Our results indicate that amoxicillin significantly increased the MIC values of several tested antibiotics, except for oxytetracycline and florfenicol. In the case of cefotaxime, this increase was observed in all classes. A total of 44 antimicrobial resistance genes were identified in all samples. Chromosomal point mutations, particularly concerning cefotaxime, revealed numerous complex mutations, deletions, insertions, and single nucleotide polymorphisms (SNPs) that were not experienced in the case of amoxicillin. The findings suggest that, regarding amoxicillin, the point mutation of the acrB gene could explain the observed MIC value increases due to the heightened activity of the acrAB-tolC efflux pump system. However, under the influence of cefotaxime, more intricate processes occurred, including complex amino acid substitutions in the ampC gene promoter region, increased enzyme production induced by amino acid substitutions and SNPs, as well as mutations in the acrR and robA repressor genes that heightened the activity of the acrAB-tolC efflux pump system. These changes may contribute to the significant MIC increases observed for all tested antibiotics. The results underscore the importance of understanding cross-resistance development between individual drugs when choosing clinical alternative drugs. The point mutations in the mdtB and emrR genes may also contribute to the increased activity of the mdtABC-tolC and emrAB-tolC pump systems against all tested antibiotics. The exceptionally high mutation rate induced by cephalosporins justifies further investigations to clarify the exact mechanism behind.

Background Selection From Unlinked Sites Causes Nonindependent Evolution of Deleterious Mutations.

Background selection describes the reduction in neutral diversity caused by selection against deleterious alleles at other loci. It is typically assumed that the purging of deleterious alleles affects linked neutral variants, and indeed simulations typically only treat a genomic window. However, background selection at unlinked loci also depresses neutral diversity. In agreement with previous analytical approximations, in our simulations of a human-like genome with a realistically high genome-wide deleterious mutation rate, the effects of unlinked background selection exceed those of linked background selection. Background selection reduces neutral genetic diversity by a factor that is independent of census population size. Outside of genic regions, the strength of background selection increases with the mean selection coefficient, contradicting the linked theory but in agreement with the unlinked theory. Neutral diversity within genic regions is fairly independent of the strength of selection. Deleterious genetic load among haploid individuals is underdispersed, indicating nonindependent evolution of deleterious mutations. Empirical evidence for underdispersion was previously interpreted as evidence for global epistasis, but we recover it from a non-epistatic model.

Abstract IA002: Management of newly diagnosed endometrial cancers and molecular classification

Abstract In the past 50 years, the treatment of this cancer has evolved from a regimen of preoperative radiation therapy followed by hysterectomy, to individualized management consisting of surgical treatment with or without additional therapy depending on whether various risk factors are identified. After the diagnosis of endometrial carcinoma has been histologically confirmed, uterine and extrauterine risk factors affect the probability of retroperitoneal lymph node involvement and subsequent survival. Surgical staging is a critical component for the proper diagnosis and management of all endometrial carcinomas. FIGO stage is the most powerful predictor of prognosis. Surgical staging is feasible by minimally invasive techniques. Patients with significant medical comorbidities who are not acceptable candidates for surgery may be managed by alternative means. Women with EC are diagnosed under the age of 40 may be eligibile for nonoperative management if there is no clear evidence of myometrial invasion. As the contemporary management of EC continues to evolve, and the indications for lymph node dissection remain controversial. Minimally invasive management of EC has increasingly been integrated into standard practice. In 2006, The Cancer Genome Atlas (TCGA) began with studies in glioblastoma and high-grade serous ovarian cancer. The National Institutes of Health committed major resources to TCGA to collect and characterize the genomic landscape of more than 20 cancer types. In 2013, TCGA published findings for endometrial cancers. Only serous and endometrioid subtypes were accrued in the endometrial cancer project. TCGA has reclassified endometrioid and serous endometrial cancers in four broad categories using comprehensive genomic approaches. One-third of the cancers is characterized by microsatellite instability (MSI) and associated hyper-mutation. The cancers without MSI can be further separated into two groups. Serous cases with extensive DNA copy number alterations and genomic instability comprise one group, and endometrioid cases with few or only focal copy number alterations comprise the other. A smaller fourth sub-group contains samples with very high mutation rates and is characterized by mutations in POLE. Molecular features can be distinctly overlaid onto these four sub-classifications, and each have important clinical implications that are currently being used in both clinical practice and clinical trial design. In this educational presentation, we will discuss the management of newly diagnosed endometrial cancers, review the molecular classification of endometrial cancers, and identify approaches that incorporate these findings into clinical care and clinical trial design. Citation Format: Douglas A. Levine. Management of newly diagnosed endometrial cancers and molecular classification [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA002.

Targeted next-generation sequencing of 491 lung cancers in clinical practice: Implications for future detection strategy and targeted therapy

Although lung cancer remains the most common cause of global cancer-related mortality, the identification of oncogenic driver alterations and the development of targeted drugs has dramatically altered the therapeutic landscape. In this retrospective study, we found that 97.7% samples carried at least one mutation in the 25 genes tested in our cohort. 53.6% samples were positive for EGFR mutations, followed by TP53 (41.1%), KRAS (11.8%), ERBB2 (4.3%). EGFR mutations were mainly found in female adenocarcinomas, while TP53 was mainly found in male non-adenocarcinomas. Significant differences can be found in the mutation rate of EGFR (60.9% vs 11.9%), KRAS (12.2% vs 25.0%), STK11 (1.5% vs 11.9%), FGFR3 (2.4% vs 0.0%) and ERBB4 (1.2% vs 6.1%) between adenocarcinoma in our cohort and TCGA-LUAD data (all p < 0.001). What's more, we found that the mutation of EGFR increased significantly from adenocarcinomas in situ (AIS, 21.4%) to microinvasive adenocarcinomas (MIA, 52.4%) and invasive adenocarcinomas (IA, 61.1%), while the mutation of ERBB2 dropped markedly from AIS (21.4%) to MIA (9.5%) and IA (4.1%). At last, comparations between targeted NGS and ARMS-based single gene test in the detection of EGFR showed a 94.6% consistence. In conclusion, targeted NGS can provide a comprehensive mutational profile of lung cancer. Considering the high mutation rate of EGFR in NSCLC of Asian populations, a specialized detection strategy should be conducted.

A pan-cancer multi-omics analysis of lactylation genes associated with tumor microenvironment and cancer development

BackgroundLactylation is a significant post-translational modification bridging the gap between cancer epigenetics and metabolic reprogramming. However, the association between lactylation and prognosis, tumor microenvironment (TME), and response to drug therapy in various cancers remains unclear. MethodsFirst, the expression, prognostic value, and genetic and epigenetic alterations of lactylation genes were systematically explored in a pan-cancer manner. Lactylation scores were derived for each tumor using the single-sample gene set enrichment analysis (ssGSEA) algorithm. The correlation of lactylation scores with clinical features, prognosis, and TME was assessed by integrating multiple computational methods. In addition, GSE135222 data was used to assess the efficacy of lactylation scores in predicting immunotherapy outcomes. The expression of lactylation genes in breast cancers and gliomas were verified by RNA-sequencing. ResultsLactylation genes were significantly upregulated in most cancer types. CREBBP and EP300 exhibited high mutation rates in pan-cancer analysis. The prognostic impact of the lactylation score varied by tumor type, and lactylation score was a protective factor for KIRC, ACC, READ, LGG, and UVM, and a risk factor for CHOL, DLBC, LAML, and OV. In addition, a high lactylation score was associated with cold TME. The infiltration levels of CD8+ T, γδT, natural killer T cell (NKT), and NK cells were lower in tumors with higher lactylation scores. Finally, immunotherapy efficacy was worse in patients with high lactylation scores than other types. ConclusionLactylation genes are involved in malignancy formation. Lactylation score serves as a promising biomarker for predicting patient prognosis and immunotherapy efficacy.

Modeling viral evolution: A novel SIRSVIDE framework with application to SARS-CoV-2 dynamics

Understanding evolutionary trends in emerging viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is crucial for effective public health management and response. Nonetheless, extensive debates have arisen concerning viral evolutionary trends, particularly the interplay between transmissibility, pathogenicity, and immune escape. In this context, we have developed a novel computational model named SIRSVIDE (Susceptible-Infected-Recovered-Susceptible-Variation-Immune Decay-Immune Escape) to simulate the transmission and evolutionary dynamics of viral populations. Our simulation results indicate that under conditions of high mutation rates, elevated transmission rates, and larger susceptible host populations, viral populations exhibit prolonged increases in transmissibility and immune escape, accompanied by reductions in pathogenicity and noticeable short-term fluctuations. However, when the total susceptible population size and mutation rate decrease, substantial uncertainty in the evolutionary trends of viral populations becomes apparent. In summary, the SIRSVIDE model establishes a comprehensive framework for generating both short- and long-term viral epidemiological and evolutionary dynamics. The simulation outcomes align with existing evidence indicating that SARS-CoV-2 is undergoing selection for heightened transmissibility, decreased pathogenicity, and enhanced immune escape. Furthermore, the model sheds light on the possible evolutionary dynamics of other viruses.

Geometric analysis of SARS-CoV-2 variants

SARS-CoV-2 as a severe respiratory disease has been prevalent around the world since its first discovery in 2019.As a single-stranded RNA virus, its high mutation rate makes its variants manifold and enables some of them to have high pathogenicity, such as Omicron variant, the most prevalent virus now. Research on the relationship of these SARS-CoV-2 variants, especially exploring their difference is a hot issue. In this study, we constructed a geometric space to represent all SARS-CoV-2 sequences of different variants. An alignment-free method: natural vector method was utilized to establish genome space. The genome space of SARS-CoV-2 was constructed based on the 24-dimensional natural vector and the appropriate metric was determined through performing phylogenetic analysises. Phylogenetic trees of different lineages constructed under the selected natural vector and metric coincided with the lineage naming standards, which means lineages with same alphabetical prefix cluster in phylogenetic trees. Furthermore, the relationships between the various GISAID clades as depicted by the natural graph primarily matched the description provided in the GISAID clade naming.The validity of our geometric space was demonstrated by these phylogenetic analysis results. So in this research, we constructed a geometry space for the genomes of the novel coronavirus SARS-CoV-2, which allows us to compare the different variants. Our geometric space is valuable for resolving the issues insides the virus.

Enhanced selective discrimination of point-mutated viral RNA through false amplification regulatory direct insertion in rolling circle amplification

Coronaviruses are single-stranded RNA viruses with high mutation rates. Although a diagnostic method for coronaviruses has been developed, variants appear rapidly. Low test accuracy owing to single-point mutations is one of the main factors in the failure to prevent the early spread of coronavirus infection. Although reverse transcription-quantitative polymerase chain reaction can detect coronavirus infection, it cannot exclude the possibility of false positives, and an additional multiplexing kit is needed to discriminate single nucleotide polymorphism (SNP) variants. Therefore, in this study, we introduced a new nucleic acid amplification method to determine whether an infected person has a SNP mutation using a lateral flow assay (LFA) as a point-of-care test. Unlike traditional DNA amplification methods, direct insertion into rolling circle amplification amplifies the target genes without false amplification. After SNP-selective nucleic acid amplification, nuclease enzymes are used to make double-stranded DNA fragments that the LFA can detect, where specific mismatched DNA is found and cleaved to show different signals when a SNP-type is present. Therefore, wild- and SNP-type variants can be selectively detected. In this study, the limit of detection was 400 aM for viral RNA, and we successfully identified a dominant SNP variant selectively. Clinical tests were also conducted.

Real-time evaluation of signal accuracy in wastewater surveillance of pathogens with high rates of mutation

Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation.

Clinical impact of pleural fluid carcinoembryonic antigen on therapeutic strategy and efficacy in lung adenocarcinoma patients with malignant pleural effusion.

Epidermal growth factor receptor (EGFR) mutation is important in determining the treatment strategy for advanced lung cancer patients with malignant pleural effusion (MPE). Contrary to serum carcinoembryonic antigen (S-CEA) levels, the associations between pleural fluid CEA (PF-CEA) levels and EGFR mutation status as well as between PF-CEA levels and treatment efficacy have rarely been investigated in lung adenocarcinoma patients with MPE. This retrospective study enrolled lung adenocarcinoma patients with MPE and available PF-CEA levels and EGFR mutation results. The patients were categorized based on PF-CEA levels: < 10 ng/mL, 10-100 ng/mL, 100-500 ng/mL, and ≥ 500 ng/mL. The association between PF-CEA levels and EGFR mutation status as well as their therapeutic impact on overall survival was compared among the four groups. This study included 188 patients. PF-CEA level was found to be an independent predictor of EGFR mutation but not S-CEA level. The EGFR mutation rates were higher as the PF-CEA levels increased, regardless of cytology results or sample types. Among EGFR-mutant lung adenocarcinoma patients receiving EGFR-tyrosine kinase inhibitor (TKI) treatment, those with high PF-CEA levels had significantly better survival outcomes than those with low PF-CEA levels. High PF-CEA levels were associated with high EGFR mutation rate and may lead to a favorable clinical outcome of EGFR-TKI treatment in EGFR-mutant lung adenocarcinoma patients with MPE. These findings highlight the importance of actively investigating EGFR mutation detection in patients with suspected MPE and elevated PF-CEA levels despite negative cytology results.

High germline mutation rates, but not extreme population outbreaks, influence genetic diversity in a keystone coral predator.

Lewontin's paradox, the observation that levels of genetic diversity (π) do not scale linearly with census population size (Nc) variation, is an evolutionary conundrum. The most extreme mismatches between π and Nc are found for highly abundant marine invertebrates. Yet, the influences of new mutations on π relative to extrinsic processes such as Nc fluctuations are unknown. Here, we provide the first germline mutation rate (μ) estimate for a marine invertebrate in corallivorous crown-of-thorns sea stars (Acanthaster cf. solaris). We use high-coverage whole-genome sequencing of 14 parent-offspring trios alongside empirical estimates of Nc in Australia's Great Barrier Reef to jointly examine the determinants of π in populations undergoing extreme Nc fluctuations. The A. cf. solaris mean μ was 9.13 x 10-09 mutations per-site per-generation (95% CI: 6.51 x 10-09 to 1.18 x 10-08), exceeding estimates for other invertebrates and showing greater concordance with vertebrate mutation rates. Lower-than-expected Ne (~70,000-180,000) and low Ne/Nc values (0.0047-0.048) indicated weak influences of population outbreaks on long-term π. Our findings are consistent with elevated μ evolving in response to reduced Ne and generation time length, with important implications for explaining high mutational loads and the determinants of genetic diversity in marine invertebrate taxa.

Distinctive development of embryo and endosperm caused by male gametes irradiated with carbon-ion beam

Abstract Key message In Cyrtanthus mackenii, development of embryo and endosperm were differentially affected by fertilization of male gametes with DNA damage and mutations. Abstract Pollen irradiation with ionizing radiations has been applied in plant breeding and genetic research, and haploid plant induction has mainly been performed by male inactivation with high-dose irradiation. However, the fertilization process of irradiated male gametes and the early development of embryo and endosperm have not received much attention. Heavy-ion beams, a type of radiation, have been widely applied as effective mutagens for plants and show a high mutation rate even at low-dose irradiation. In this study, we analyzed the effects of male gametes of Cyrtanthus mackenii irradiated with a carbon-ion beam at low doses on fertilization. In immature seeds derived from the pollination of irradiated pollen grains, two types of embryo sacs were observed: embryo sac with a normally developed embryo and endosperm and embryo sac with an egg cell or an undivided zygote and an endosperm. Abnormalities in chromosome segregation, such as chromosomal bridges, were observed only in the endosperm nuclei, irrespective of the presence or absence of embryogenesis. Therefore, in Cyrtanthus, embryogenesis is strongly affected by DNA damage or mutations in male gametes. Moreover, various DNA contents were detected in the embryo and endosperm nuclei, and endoreduplication may have occurred in the endosperm nuclei. As carbon-ion irradiation causes chromosomal rearrangements even at low doses, pollen irradiation can be an interesting tool for studying double fertilization and mutation heritability.

Integrating cfDNA liquid biopsy and organoid-based drug screening reveals PI3K signaling as a promising therapeutic target in colorectal cancer

BackgroundThe current precision medicine relies on biomarkers, which are mainly obtained through next-generation sequencing (NGS). However, this model failed to find effective drugs for most cancer patients. This study tried to combine liquid biopsy with functional drug tests using organoid models to find potential drugs for cancer patients.MethodsColorectal cancer (CRC) patients were prospectively enrolled and blood samples were collected from patients before the start of treatment. Targeted deep sequencing of cfDNA samples was performed using a 14-gene panel. Gastrointestinal (GI) cancer organoids were established and PI3K and mTOR inhibitors were evaluated on organoid models.ResultsA total of 195 mutations were detected across 58 cfDNA samples. The most frequently mutated genes were KRAS, TP53, PIK3CA, and BRAF, all of which exhibited higher mutation rates than tissue biopsy. Although 81% of variants had an allele frequency of less than 1%, certain mutations in KRAS, TP53, and SMAD4 had high allele frequencies exceeding 10%. Notably, among the seven patients with high allele frequency mutations, six had metastatic tumors, indicating that a high allele frequency of ctDNA could potentially serve as a biomarker of later-stage cancer. A high rate of PIK3CA mutation (31 out of 67, or 46.3%) was discovered in CRC patients, suggesting possible tumor progression mechanisms and targeted therapy opportunities. To evaluate the value of anti PI3K strategy in GI cancer, different lines of GI cancer organoids were established. The organoids recapitulated the morphologies of the original tumors. Organoids were generally insensitive to PI3K inhibitors. However, CRC-3 and GC-4 showed response to mTOR inhibitor Everolimus, and GC-3 was sensitive to PI3Kδ inhibitor Idelalisib. The CRC organoid with a PIK3CA mutation showed greater sensitivity to the PI3K inhibitor Alpelisib than wildtype organoids, suggesting potential treatment options for the corresponding patients.ConclusionLiquid biopsy holds significant promise for improving precision treatment and tumor prognosis in colorectal cancer patients. The combination of biomarker-based drug prediction with organoid-based functional drug sensitivity assay may lead to more effective cancer treatment.

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022.

Avian influenza viruses are able to cross the species barrier between birds and mammals because of their high genetic diversity and mutation rate. Using formalin-fixed paraffin-embedded tissues, we were able to investigate a Tibetan black bear's infection by a high pathogenicity H5N1 avian influenza virus at the molecular, phylogenetic, and histological levels. Our results highlight the importance of virological surveillance programs in mammals and the importance of raising awareness among veterinarians and zookeepers of the clinical presentations associated with H5Nx virus infection in mammals.

Modeling and Analysis of HIV-1 Pol Polyprotein as a Case Study for Predicting Large Polyprotein Structures.

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.

Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.