Search Results (464)

The evolution of robust DNA repair mechanisms was a prerequisite for the conquest of land by plants, a transition that exposed them to harsh new environmental stressors. The poly (ADP-ribose) polymerase (PARP) family is central to this adaptation, as it orchestrates DNA repair and stress signaling pathways essential for coping with the elevated UV radiation and desiccation of terrestrial environments. Yet its early evolutionary origins are unknown. Here, we present a comprehensive reconstruction of the PARP family’s history across the plant kingdom. Our phylogenomic analysis reveals that PARP evolution ignited during the bryophyte radiation, expanding from a single ancestral algal gene into three distinct subfamilies (PARP1, PARP2, and PARP3). This diversification was driven by structural innovations in DNA-binding domains and a rewiring of transcriptional networks to respond to terrestrial challenges. We provide direct experimental support for this hypothesis through functional analysis of PARPs from the extremotolerant moss Syntrichia caninervis. We show that its PARP proteins provide multifaceted protection against UV radiation, heat, and genotoxic agents, and that recently duplicated PARP2 genes are already diverging in function. Our work pinpoints the molecular adaptations in a key DNA repair family that enabled the greening of Earth and uncovers novel genetic targets for enhancing crop resilience. Full article

SARS-CoV-2 continues to evolve into immune-evasive variants, and although vaccination remains the cornerstone of prevention, the search for antiviral molecules targeting conserved viral enzymes remains essential. The RNA-dependent RNA polymerase (NSP12) is a central component of coronavirus replication, and natural polyphenols have been recurrently proposed as modulators of viral polymerases. Among these compounds, Fisetin has been reported to interact with multiple viral and cellular pathways, yet its direct antiviral activity against SARS-CoV-2 remained largely unexplored. Here, we first analyzed the interaction of Fisetin with the catalytic and NiRAN domains of NSP12 using molecular docking and molecular dynamics simulations, revealing stable and energetically favorable binding throughout a 100 ns simulation. Previous biochemical reports have shown that Fisetin inhibits the recombinant SARS-CoV-2 RdRp, supporting its potential to engage the polymerase. We then evaluated its antiviral activity in human A549 lung epithelial cells infected with the Omicron JN.1 variant. We observed a clear dose-dependent reduction in viral infection, achieving up to 91.9% inhibition at 3 μM while maintaining acceptable cell viability. In addition, Fisetin displayed a selectivity index superior to that of Lopinavir, the positive antiviral control used in this study. Altogether, our findings demonstrate that Fisetin possesses reproducible antiviral activity in a physiologically relevant human lung model and support its role as a natural scaffold for the rational development of polymerase-targeting antivirals against emerging SARS-CoV-2 variants. Full article

In sturgeon aquaculture, all-female production is desirable due to the high value of caviar. Genetic sexing and the production of WW superfemales are important steps toward achieving this. In this study, we identified the WSR and ZSR primers for amplification of W- and Z-specific regions, respectively. WSR primers were designed on the gene W-linked RT RNase H-like domain containing protein (rnhW). The polymerase chain reaction (PCR) bands were obtained with the WSR primer only in phenotypic female sturgeons, indicating that stable genetic sexing was achieved in most species, including those captured around Hokkaido. Moreover, rnhW showed female-specific expression in the gonads during early sex differentiation in kaluga and Amur sturgeon. ZSR primers were developed from the orofacial cleft 1 candidate gene 1 protein homolog. Clear and distinct gel band patterns for ZZ, ZW, and WW genotypes were obtained using WSR and ZSR primers, consistent with genotypic estimations by quantitative PCR. This consistency confirmed the presence of WW superfemales among offspring produced by fertilizing ZW females with ZW pseudomales masculinized using 17α-methyltestosterone. Our findings provide new insights into the mechanisms of sex determination and differentiation in sturgeons, bringing the establishment of an all-female production system within reach. Full article

Cystic fibrosis (CF) is a rare genetic disease caused by pathogenic variants in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, with wide clinical variability influence not only by the CFTR genotype but also by environmental and modifier genes such as ADAM33 (A Disintegrin and Metalloproteinase Domain 33). The rs2280091 variant in ADAM33 may affect lung function and contribute to differences in disease severity. This study investigated the association between this genetic variant and lung function in CF patients. This cross-sectional study included 55 CF patients from a Brazilian center, with diagnosis confirmed by sweat testing and CFTR genotyping. Pulmonary function was evaluated by spirometry before and after bronchodilator (BD) administration according to the American Thoracic Society/European Respiratory Society guidelines, analyzing Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), FEV1/FVC ratio, Forced Expiratory Flow at 25%, 50%, and 75% of FVC (FEF25%, FEF50%, and FEF75%), mean Forced Expiratory Flow between 25% and 75% of FVC (FEF25–75%), and Maximal Expiratory Forced Flow (MEF). The ADAM33 rs2280091 variant was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and statistical analyses included Kruskal–Wallis and Mann–Whitney tests, chi-square (χ²) tests, and calculation of odds ratios (ORs) with 95% confidence intervals (95% CI). The study included 55 CF patients, predominantly female (96.4%) and Caucasian (52.7%), with a median age of 17 years. CFTR genotyping revealed F508del/F508del as the most common genotype (52.7%). Analysis of the ADAM33 rs2280091 variant demonstrated that the AA genotype was most frequent in both CF patients (69.1%) and healthy controls (78.6%). Notably, the GG genotype was significantly enriched in CF patients (18.2%) compared with the controls (0.02%), yielding an odds ratio of 12.06 (95% CI: 4.86–29.91), while the G allele was also associated with increased disease risk (24.5% vs. 11.6%). Pulmonary function assessment indicated that carriers of the GG genotype or G allele had higher Forced Expiratory Flow parameters (FEF25%, FEF50%, FEF25–75%, and MEF) and improved BD responsiveness, suggesting a potential modulatory role of ADAM33 in peripheral airway function in CF. The G allele of the ADAM33 rs2280091 variant was more frequent among recruited CF patients and associated with improved peripheral airway function and BD response. These findings may reflect a survivor effect, in which carriers of this allele are more likely to reach clinical follow-up and recruitment rather than indicating a direct association with increased disease risk. Full article

Background: The minimized pan-coronavirus (CoV) vaccine-1 developed by our laboratory contained pDNA sequences of feline coronavirus serotype-1 (FCoV1) and SARS-CoV2 (SCoV2) spike B-cell epitopes plus FCoV/SCoV2-conserved, CoV-specific polymerase cytotoxic T-lymphocyte (CTL) epitopes formulated in lipid nanoparticle (LNP). Only FCoV2 infects feline cell lines needed for developing native challenge inoculum that causes feline infectious peritonitis (FIP). Hence, Pilot Study 1 evaluated the therapeutic efficacy and safety of the pan-CoV vaccine-1 in feline immunodeficiency virus (FIV)-infected cats, with or without FCoV1 coinfection. Pilot Study 2 evaluated the cross-protective effect of pan-CoV vaccines in specific-pathogen-free (SPF) cats against intranasal challenge with FIP virus serotype 2 (FIPV2). Methods: In Study 1, we vaccinated two FIV-infected cats (one negative and another positive for FCoV1 coinfection) intramuscularly twice with CTL epitopes-LNP vaccine and later twice with pan-CoV vaccine-1. Controls included two unvaccinated FIV-infected cats with or without FCoV1 coinfection. Study 2 assessed the sequential vaccinations of three pan-CoV vaccines in four SPF cats. The first two vaccinations were with pan-CoV vaccine-2, followed by pan-CoV vaccine-3 (twice), and lastly with pan-CoV vaccine-1 (once). Three SPF controls included two cats immunized with LNP and one lacking any immunization. Pan-CoV vaccine-2 contained pDNAs with modified FCoV1/SCoV2 B-cell epitopes plus CTL epitopes in LNP. Pan-CoV vaccine-3 contained only pDNAs with FCoV1 B-cell epitopes plus CTL epitopes in LNP. Results: Study 1 demonstrated no adverse effect with 25 μg and 50 μg CTL epitopes-LNP vaccine and 50 μg pan-CoV vaccine-1. However, 100 μg pan-CoV vaccine-1 caused fever 24 h later, which was resolved by a single Meloxicam treatment. Both vaccinees developed cross-FCoV2 neutralizing antibodies (XNAbs), immunoblot binding antibodies (bAbs) to FCoV1 receptor-binding domain (RBD), and T-cell responses to FCoV1 RBD, whereas one vaccinee also developed bAbs to SCoV2 RBD. Study 2 demonstrated no adverse effects after each vaccination. Three vaccinees developed low-titer XNAbs and bAbs to FCoV2 spike-2 by the fourth vaccination. Upon challenge, all cats developed FCoV2 NAbs and bAbs to FCoV2 nucleocapsid and RBD. High vaccine-induced T-cell responses to FCoV1 RBD and T-cell mitogen responses declined with an increase in responses to FCoV2 RBD at three weeks post-challenge. Two of the three controls died from FIP, whereas one vaccinee, with the lowest vaccine-induced immunity, died from skin vasculitis lesions and detection of FIPV2 infection by semi-nested RT-snPCR in feces. Conclusions: In Pilot Study 1, the pan-CoV vaccine-LNP dose of 50 μg had no adverse effects, but adverse effects were observed at 100 μg dose. In Pilot Study 2, the FCoV1-based B-cell vaccine(s) induced low levels of XNAbs against FIPV2 and delayed challenge infection against high-dose FIPV2. The high-dose FIPV2 infections in the vaccinated and control cats started to clear, by single housing at 23–26 weeks post-challenge, whereas two cats in Pilot Study 1 cleared natural FCoV1 transmission by 26 weeks post-infection. Full article

Background: Perennial ryegrass (Lolium perenne L.), a widely cultivated turfgrass and forage species in Europe and North America, exhibits rapid growth and notable salt tolerance. The high-affinity potassium transporter (HKT) gene family has been implicated in salt stress responses across multiple plant species. However, whether the salt tolerance of L. perenne is closely associated with its HKT gene family remains unclear. Methods and Results: In this study, we systematically identified HKT family members in the L. perenne genome. Five HKT genes were identified and classified into three subfamilies. Among these, LpHKT1a–c exhibited canonical class I features with a conserved serine (S) residue in the P1 domain, whereas LpHKT2 belonged to class II, characterized by a glycine (G) residue in the same domain. Notably, LpHKT3 formed a distinct subfamily with a truncated structure and divergent P1/P2 domains, suggesting potential non-canonical functions. LpHKT1a likely lacked the P4 domain. Promoter analysis revealed that all five LpHKT genes contain multiple stress-related cis-acting elements. Real-time quantitative reverse transcription polymerase chain reaction results showed that LpHKT1b/c and LpHKT2 were highly expressed in both roots and leaves. Under low-concentration NaCl stress (25 mM), the expression of these three genes significantly increased by 8- to 12-fold at 6–12 h post-treatment (vs. control). Ion accumulation analysis demonstrated a rapid increase in Na⁺ levels following NaCl treatment, whereas K⁺ concentrations initially remained stable but significantly increased after 24 h. Conclusions: Combined with the cellular localization of LpHKT1c predominantly in the xylem, these findings suggest that LpHKT genes may be involved in Na⁺ and K⁺ transport in roots. This study represents the first genome-wide identification of the HKT gene family in L. perenne, providing critical insights into the molecular mechanisms underlying its salt tolerance and offering valuable genetic resources for molecular breeding aimed at enhancing stress resilience. Full article

The picornavirus 3CD protein is a precursor to the 3C main protease and the 3D RNA-dependent RNA polymerase. In addition to its functions in proteolytic processing of the virus polyprotein and cleavage of key host factors, the 3C domain interacts with cis-acting replication elements (CREs) within the viral genome to regulate replication and translation events. We investigated the molecular determinants of RNA binding to 3C using a wide range of biophysical and computational methods. These studies showed that 3C binds to a broad spectrum of RNA oligonucleotides, displaying minimal sequence and structure dependence, at least for these shorter RNAs. However, they also uncovered a novel aspect of these interactions, that is, 3C-RNA binding can induce liquid–liquid phase separation (LLPS), with 3CD–RNA interactions likewise leading to LLPS. This may be a general phenomenon for other 3C and 3C-like proteases and polyproteins incorporating 3C domains. These findings have potential implications in understanding virally induced apoptosis and the control of stress granules, which involve LLPS and include other proteins with known interactions with 3C/3CD. Full article

JC virus (JCV) replicates within the nuclei of glial cells in the human brain and causes progressive multifocal leukoencephalopathy. JCV possesses a small, circular, double-stranded DNA genome, divided into early and late protein-coding regions. The non-coding control region (NCCR) functions bidirectionally for both early and late genes, and the agnogene is located downstream of TCR and upstream of three capsid proteins in the late region. Previously, in cell culture systems, we demonstrated that these capsid proteins accumulate in intranuclear domains known as promyelocytic leukemia nuclear bodies (PML-NBs), where they assemble into virus-like particles (VLPs). To investigate the agnogene’s function, VLPs were formed in its presence or absence, and differential gene expression was analyzed using microarray technology. The results revealed altered expression of histone-modifying enzymes, including methyltransferases (EHMT1, PRMT7) and demethylases (KDM2B, KDM5C, KDM6B), as well as various kinases and phosphatases. Notably, CTDP1, which dephosphorylates the C-terminal domain of an RNA polymerase II subunit, was also differentially expressed. The changes were predominant in the presence of the agnogene. These findings indicate that the agnogene and/or its protein product likely influence epigenetic regulation associated with PML-NBs, which may influence cell cycle control. Consistently, in human brain tissue, JCV-infected glial cells displayed maintenance of a diploid chromosomal complement, likely through G2 arrest. The precise mechanism of this, however, remains to be elucidated. Full article

Bacillus subtilis DinG/XPD-like paralogues, DinG and YpvA, have been implicated in overcoming replication stress. DinG possesses a DEDD exonuclease and DNA helicase domains, whereas YpvA lacks the DEDD exonuclease domain. We report that DinG·Mg²⁺ (hereafter referred to as DinG) degrades linear single-stranded (lss) DNA with 3′→5′ polarity and binds lssDNA with higher affinity than its exonuclease-deficient mutant DinG D10A E12A. DinG’s ssDNA-dependent ATPase activity neither stimulates nor inhibits DNA degradation. When bound to the 3′-end of forked DNA, DinG destabilises and degrades the substrate; however, in the presence of ATP, DinG dissociates before reaching the duplex junction. DinG degrades the RNA strand within RNA–DNA hybrids but does not cleave lssRNA unless complexed with Mn²⁺. DinG removes genomic R-loops, as RnhC and PcrA do. DinG physically interacts with RecA and PolA and functions in the same pathway as translesion synthesis (TLS) DNA polymerases (DNAPs) to respond to both spontaneous and methyl methanesulphonate (MMS)-induced mutagenesis. DinG-mGold forms spontaneous foci at or near replication forks, which become enriched following MMS or rifampicin treatment. We propose that DinG contributes to mitigating replication stress by degrading R-loop barriers and facilitating TLS, potentially via RecA-linked mechanisms. Full article

RNA polymerases are essential enzymes that catalyze DNA transcription into RNA, vital for protein synthesis, gene expression regulation, and cellular responses. Non-template-dependent RNA polymerases, which synthesize RNA without a template, are valuable in biological research due to their flexibility in producing RNA without predefined sequences. However, their substrate polymerization mechanisms are not well understood. This study examines Poly (A) polymerase (PAP), a nucleotide transferase superfamily member, to explore its substrate selectivity using computational methods. Previous research shows PAP’s polymerization efficiency for nucleoside triphosphates (NTPs) ranks ATP > GTP > CTP > UTP, though the reasons remain unclear. Using 500 ns Gaussian accelerated molecular dynamics simulations, stability analysis, secondary structure analysis, MM-PBSA calculations, and Markov state modeling, we investigate PAP’s differential polymerization efficiencies. Results show that ATP binding enhances PAP’s structural flexibility and increases solvent-accessible surface area, likely strengthening protein–substrate or protein–solvent interactions and affinity. In contrast, polymerization of other NTPs leads to a more open conformation of PAP’s two domains, facilitating substrate dissociation from the active site. Additionally, ATP binding induces a conformational shift in residues 225–230 of the active site from a loop to an α-helix, enhancing regional rigidity and protein stability. Both ATP and GTP form additional π–π stacking interactions with PAP, further stabilizing the protein structure. This theoretical study of PAP polymerase’s substrate selectivity mechanisms aims to clarify the molecular basis of substrate recognition and selectivity in its catalytic reactions. These findings offer valuable insights for the targeted engineering and optimization of polymerases and provide robust theoretical support for developing novel polymerases for applications in drug discovery and related fields. Full article

In this study, 944 Subo Merino sheep, a high-quality fine wool breed, were selected as research subjects. The SNP typing of the FAT3 gene was performed using the Fluidigm BiomarkTM HD system, and 11 missense mutation sites were identified. The analysis of population polymorphism of single-nucleotide polymorphisms was conducted. It is noteworthy that a substantial strong linkage disequilibrium was identified between SNP 5 and SNP 6 (r² > 0.8). The association between SNPs of the FAT3 gene and wool traits showed that multiple SNPs were significantly correlated with several different wool traits (p < 0.05). Furthermore, the investigation delved into the impact of the FAT3 gene on wool fiber through the utilization of quantitative polymerase chain reaction (qPCR), which yielded findings that this gene was notably expressed in fine wool fiber (FW) (p < 0.001). To predict the subcellular localization and protein transmembrane structure of FAT3, we employed the PSORT II Prediction and TMHMM online software. It was determined that the protein contains a transmembrane domain. This study provides molecular markers for the improvement of the selection and breeding of ultrafine-wool sheep and offers experimental evidence for accelerating the genetic breeding of sheep. Full article

The research aimed to study the genome of Cryptococcus neoformans isolated from bird excreta. Thirteen isolates were cultured, colony stained, and underwent biochemical testing confirmation by nested polymerase chain reaction using ITS1-ITS4 and CN4-CN5 primers, respectively. Antifungal susceptibility testing and whole-genomic sequencing were analyzed. The results determined that all isolates were susceptible to amphotericin B (100%), fluconazole, and itraconazole (92.3%). One isolate (DOP3) showed resistance to fluconazole and itraconazole (MIC >64 and >8 µg/mL, respectively). A phylogenetic tree showed the identity of C. neoformans (serotype A). The genome of resistant (DOP3) and non-resistant isolates (DOP3.1) had 14 chromosomes. DOP3 consisted of 38 candidate antifungal resistance genes, which were the most active against azoles (14). The annotated genes in the azole group mostly were in the ATP-binding cassette transporter transmembrane superfamily. Resistance genes against FCZ were in the transcription factors (HAP2, HAP5), zinc finger (NRG1), cytochrome P450 (ERG11), and Myb-like DNA-binding domain (REB1). The most frequent resistance genes against ITZ were cytochrome P450 (ERG5 and ERG11) and a transcription factor (HAP5). DOP3.1 also consisted of 26 candidate resistance genes against azoles. Resistance genes against the azole group belong to the ABC transporter transmembrane superfamily. Resistance genes against FCZ belong to cytochrome P450 (ERG11), the zinc finger (NRG1), and the CCAAT binding transcription factor (HAP2). Resistance genes belonging to cytochrome P450 (ERG5) were found against ITZ. This research provides the first report of C. neoformans (serotype A) in zebra dove excreta, drug susceptibility to a resistant strain, and identification of resistance genes. Farm sanitation should be strictly applied, and immunocompetent people should avoid contact with zebra dove excreta. Full article

The hepatitis B virus (HBV) is a DNA virus of major public health concern whose error-prone polymerase has driven the emergence of ten distinct genotypes and a multitude of resistance-associated mutations (RAMs). Herein, we conducted a retrospective observational study analyzing 8152 hepatitis B virus (HBV) sequences from 27 regions across the Americas, retrieved from GenBank, to construct a database and examine associations among HBV genotypes/subtypes, geographic distribution, resistance-associated mutations (RAMs), and resistance to nucleos(t)ide analogs (NAs) used in the treatment of chronic infection. Following phylogenetic analysis, mutations at clinically relevant sites in the reverse transcriptase domain were identified and classified by resistance to NAs. Genotypes A (21.1% A2 and 14.7% A1) and D predominated across the retrieved database, whereas genotypes E, G, H, and I each accounted for fewer than 3% of the sequences. Among the sequences in the database, 10.6% harbored RAMs, with genotypes G, A, and H predominating in this category. The most frequently observed RAM was L180M + M204V/I, which is associated with resistance to LMV, ETV, and TBV, whereas resistance to ADV and TDF remained rare. Genotypes G and A2 were significantly associated with a higher likelihood of harboring multiple RAMs (as evaluated by logistic regression), along with an increased risk of resistance to LMV, ETV, and TBV; the opposite was true for subtype A1. Notably, genotypes H and B5 were associated with an elevated risk of TDF resistance. A comprehensive understanding of RAMs and circulating genotypes in the Americas is essential for identifying high-risk populations and establishing geographically targeted therapeutic strategies. Full article

DNA polymerase epsilon (POLe) is the leading strand replicative polymerase. POLe mutations located primarily in the proofreading domain cause replication errors and increase mutation burden in cancer cells. Consequently, POLe has been classified as a cancer driver gene. Certain POLe frameshift mutations that affect the proofreading domain are purified in cancer cells, but point mutations in other domains have also been reported. Here we use an artificial intelligence algorithm to determine what other mutations co-occur with POLe mutations in colorectal cancers. We partitioned POLe mutations into driver, passenger, and WT (no mutation), then assessed mutations in other genes in these three groups. We found that a driver POLe mutation is not likely to associate with driver mutations in other genes. Thus, driver mutations in colorectal cancers appear to purify in a manner that is independent of POLe. Mutations that affect POLe function do not necessarily increase the frequency of driver mutations in other genes. Structural analysis shows that many POLe driver mutations affect coordination of the Mg²⁺ ion in the active site. Our data show that the accumulation of colorectal cancer mutations is driven by complex factors. Full article

A novel barna-like virus was found to be associated with field-collected Afrina sporoboliae plant-parasitic nematodes. The positive-sense, single-stranded RNA genome of this virus, named Afrina barna-like virus (AfBLV), comprises 4020 nucleotides encoding four open reading frames (ORFs). ORF 1 encodes a protein product spanning a transmembrane, a peptidase, and VPg domains, whereas an overlapping ORF 2 encodes an RNA-dependent RNA polymerase (RdRP). ORF2 may be expressed via a −1 translational frameshift. In phylogenetic reconstructions, the RdRP of AfBLV was placed inside a separate clade of barna and barna-like viruses related to but distinct from the genera in the Solemoviridae and Alvernaviridae families, within the overall lineage of Sobelivirales. ORF 3 of AfBLV encodes a protein product of 206 amino acids (aa) long with homology to a putative protein encoded by a similarly positioned gene of an uncharacterized virus sequence identified previously as Barnaviridae sp. ORF 4 encodes a 161 aa protein with no significant similarities to sequences in the GenBank databases. AfBLV is the first barnavirus found in a nematode. Sequence comparisons of the AfBLV genome and genomes of other barna-like viruses suggested that a recombination event was involved in the evolution of AfBLV. Analyses of the phylogeny of RdRPs and genome organizations of barna-like and solemo-like viruses support the re-classification of Barnavirus and Dinornavirus genera as members of the Solemoviridae family. Full article