Search Results (439)

The Guinea Pig X Virus (GPXV), a newly identified gammaherpesvirus, provides an opportunity to study viral evolution and host–virus dynamics. This study characterizes the GPXV genome and investigates its phylogenetic relationships and divergence from related viruses through comparative genomic and phylogenetic analyses. Virus propagation was conducted in Vero cells, followed by genomic DNA extraction and pan-herpesvirus nested PCR. Sanger sequencing filled gaps in the initial genome assembly, and whole-genome sequencing was performed using the Illumina MiSeq platform. Phylogenetic analyses focused on ORF8 (glycoprotein B), ORF9 (DNA polymerase catalytic subunit), ORF50 (RTA: replication and transcription activator), and ORF73 (LANA: latency-associated nuclear antigen). Results showed that GPXV ORFs showed variable evolutionary relationships with other gammaherpesviruses, including divergence from primate-associated viruses and clustering with bovine and rodent viruses. In addition to phylogenetics, a comprehensive comparative analysis of protein-coding genes between GPXV and the previously described Guinea Pig Herpes-Like Virus (GPHLV) revealed divergence. Twenty-four non-ORF genomic features were unique to GPXV, while 62 shared ORFs exhibited low to high sequence divergence. These findings highlight GPXV’s distinct evolutionary trajectory and its potential role as a model for studying host-specific adaptations and gammaherpesvirus diversity. Full article

Polymerase-coupled nanopore sequencing requires DNA polymerases with strong strand displacement activity and high processivity to sustain continuous signal generation. In this study, we characterized two novel B family DNA polymerases, SRHS and BBum, isolated from Bacillus phages SRT01hs and BeachBum, respectively. Both enzymes exhibited robust strand displacement, 3′→5′ exonuclease activity, and maintained processivity under diverse reaction conditions, including across a broad temperature range (10–45 °C) and in the presence of multiple divalent metal cofactors (Mg²⁺, Mn²⁺, Fe²⁺), comparable to the well-characterized Phi29 polymerase. Through biochemical analysis of mutants designed using AlphaFold3-predicted structural models, we identified key residues (G96, M97, D486 in SRHS; S97, M98, A493 in BBum) that modulated exonuclease activity, substrate specificity and metal ion utilization. Engineered variants SRHS_F and BBum_Pro_L efficiently incorporated unnatural nucleotides in the presence of Mg²⁺—a function not observed in Phi29 and other wild-type strand-displacing B family polymerases. These combined biochemical features highlight SRHS and BBum as promising enzymatic scaffolds for nanopore-based long-read sequencing platforms. Full article

Glanders is a highly contagious and often fatal zoonotic disease of equids caused by Burkholderia mallei, a pathogen of significant concern due to its potential for bioterrorism. In Brazil, glanders remains endemic, particularly among working equids in the Northeast region. Diagnostic confirmation typically involves serology, culture, and polymerase chain reaction (PCR), although false-negative PCR results have been increasingly reported. This study aimed to evaluate the diagnostic performance and analytical sensitivity of four B. mallei-specific PCR primer sets using samples from 30 seropositive equids. Microbiological cultures were obtained from various organs and swabs, followed by PCR targeting four genomic regions: fliP-IS407A(a), fliP-IS407A(b), Burk457, and Bm17. All animals were confirmed positive for B. mallei via culture, but PCR detection rates varied significantly across primer sets. The fliP-IS407A(b) primer set showed the highest sensitivity, detecting 86% of samples, while the WOAH-recommended fliP-IS407A(a) set had the lowest performance (13.4%). Analytical sensitivity assays confirmed that fliP-IS407A(b) and Bm17 primers detected DNA concentrations as low as 0.007 ng, outperforming the others. These findings suggest that certain widely used primer sets may lack sufficient sensitivity for reliable detection of B. mallei, especially in chronically infected animals with low bacterial loads. The study underscores the need for ongoing validation of molecular diagnostics to improve the detection and control of glanders in endemic regions. Full article

To date, five herpesviruses have been identified in Leporidae (LeHV-1, LeHV-2, LeHV-3, LeHV-4, and LeHV-5). Two of these have been shown to infect the European rabbit (Oryctolagus cuniculus), causing either asymptomatic infection (LeHV-2, a gammaherpesvirus) or virulent disease (LeHV-4, an alphaherpesvirus). Unfortunately, apart from LeHV-4, for which complete genome sequences are available, molecular data on leporid herpesviruses are extremely limited, with no sequences available in public databases for LeHV-1 and LeHV-3, and only a few short sequences for LeHV-2 and LeHV-5. In this study, we investigated the presence of herpesviruses in biological samples from wild rabbits (n = 34) found dead in the field during 2024. A pan-herpesvirus nested PCR directed to the herpesviral DNA polymerase gene was used for screening. Positive samples (n = 14, 41.17%) were further investigated by sequencing analysis of a longer region of the DNA polymerase gene, as well as the glycoprotein B gene and the terminase gene. Blastn analysis of the amplicons revealed the highest similarity to gammaherpesvirus. Phylogenetic analyses based on glycoprotein B, DNA polymerase, and concatenated amino acid sequences consistently placed the newly identified LeHV-6 in close proximity to LeHV-5. Both viruses form a well-supported clade within the Gammaherpesvirinae, clustering with rodent-associated herpesviruses, such as Murine herpesvirus, MuHV-4, and A. sylvaticus rhadinovirus 1. Considering the species susceptibility and the nucleotide similarities with the five previously described leporid herpesviruses, we conclude that a new rabbit gammaherpesvirus has been identified, which we propose to name LeHV-6. Full article

Background: Xanthomonas oryzae pv. oryzae (Xoo) causes rice leaf blight (BLB) and poses a major threat to global rice production. In rice production, tetramycin agents provide good control of rice leaf blight, while the standardization of the reference genes of Xoo under tetramycin stress has not been reported. The aim of this study was to identify the most stable reference genes for quantitative PCR analysis of Xoo under tetramycin stress. Methods: Six candidate reference genes, gyrB (RNA polymerase β gene), GADPH (glyceraldehyde-3-phosphate dehydrogenase gene), recA (recombinase A gene), gyrA (citrate synthase encoding gene), dnaK (molecular chaperone protein gene), and 16S rRNA (16S ribosomal RNA gene) were selected and their expression stability was assessed under tetramycin stress conditions using real-time quantitative PCR (qRT-PCR). GeNorm, NormFinder, BestKeeper and RefFinder were used to assess the expression stability, the relative expression values of the eight genes involved QS (Quorum sensing) pathway under tetramycin stress were used to validate by the rpf (regulation of pathogenic factors) gene family. Results: 16S rRNA expression was most stable under tetracycline stress, whereas GADPH was the least. The rpf gene family showed a highly stable expression level, confirming the reliability of 16S r RNA as a reference gene in the study of Xoo under tetramycin stress. Conclusions: 16S rRNA was identified as the best reference gene for Xoo gene expression analysis under tetramycin stress. It provides a reliable support for the molecular research on the control strategy of rice BLB. Full article

Background/Objectives: Breast cancer (BC) is a multifactorial disease, with genetic alterations in cell proliferation and migration pathways being significant risk factors. This study examines the association between three variants in the BIRC5 gene (rs8073069, rs17878467, and rs9904341) and breast cancer (BC) susceptibility. Methods: Peripheral blood DNA samples were collected from 423 women (221 BC patients and 202 healthy controls). Genotyping was performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methodology. Associations were calculated using odds ratios (OR), with p-values adjusted by the Bonferroni test (significance at p ≤ 0.016). In silico analyses were conducted to predict the functional impact of the analyzed variants. Results: Patients carrying the C/C genotype for the rs8073069 variant showed increased susceptibility to BC with early TNM (tumor-node-metastasis classification) stage and Luminal A subtype (OR > 2.00; p ≤ 0.004). For the rs17878467 variant, patients with the C/T or T/T genotype exhibited a higher susceptibility to developing breast cancer (BC), particularly at early TNM stages or with a histological lobular type (OR > 2.00; p ≤ 0.012). Regarding the rs9904341 variant, patients with the G/C or C/C genotype had a higher susceptibility to breast cancer. Notably, G/C genotype carriers with Luminal A and B subtypes, and C/C genotype carriers who had TNM stages II and III, and Luminal A, Luminal B, and HER2 subtypes demonstrated increased risk (OR > 2.00; p ≤ 0.009). The C-T-C haplotype (rs8073069–rs17878467–rs9904341) was significantly associated with BC (OR = 4.20; 95% CI = 2.38–7.41; p ≤ 0.001). In silico analysis using CADD indicated a low probability of deleterious effects. Conclusions: The results suggest that the rs8073069, rs17878467, and rs9904341 variants in BIRC5 have a significant influence on breast cancer susceptibility. Full article

The Base Excision Repair (BER) pathway involves a highly coordinated series of protein–protein interactions that facilitate the recognition, excision, and repair of damaged bases. Key enzymes such as DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), polynucleotide kinase-phosphatase (PNKP), DNA polymerase b (Pol β), ligase IIIα (LigIIIα), poly (ADP-ribose) polymerases PARP1 and PARP2, and X-ray repair cross-complementing protein 1 (XRCC1) catalyze BER in a tightly regulated molecular network. These interactions ensure the seamless handoff of DNA intermediates between the core enzymes of the BER pathway. Understanding the details of protein–protein interactions in BER provides valuable insights into the molecular underpinnings of DNA repair processes. In this review, we focus on protein–protein interactions between the components of the single-nucleotide BER (SN-BER) pathway and other proteins that interact with BER components and regulate the coordination of the pathway. We also briefly discuss the interactions of other proteins that interact with the components of SN-BER based on functional evidence. Full article

The control of gene expression by cis-regulatory DNA sequences is a conserved genomic feature. The maize booster1 gene (b1) is a naturally occurring locus that serves as a mechanistic model for the control of gene expression from a distal cis element and a form of allelic interactions called paramutation. Two epi-alleles of b1 produce distinct pigmentation phenotypes correlated with transcriptional enhancement and the silencing of b1. These transcriptional dynamics depend on a hepta-tandem repeat sequence located 100 kb upstream of the b1 locus. In the heterozygous condition, the B′ epi-allele paramutates B-I, heritably converting the B-I epi-allele to the epigenetic state and expression level of B′, producing lightly pigmented plants. To identify b1TR-associated proteins, we used a targeted chromatin immunoprecipitation approach with a stably integrated transgenic b1TR locus. Applying a conservative filtering strategy, we detected several expected factors, including RNA Polymerase II, as well as the novel putative DNA-binding proteins ZAG4 and DDT4. ZAG4 and DDT4 activated GAL expression using b1TR as bait in yeast one-hybrid, supporting their potential interaction with this sequence. The identification of proteins uniquely associated with the UAS::b1TR chromatin provides insight into potential b1 regulatory factors and offers a foundation for future studies to investigate their roles in gene regulation. Full article

Background: The identification of body fluids collected from crime scenes is crucial for determining the type and nature of assaults and for advancing the resolution of crimes. Objectives: The primary aim of this study was to investigate tissue-specific DNA methylation markers that can effectively distinguish buccal samples from blood, semen, and vaginal epithelial tissue. Methods: We screened various markers and selected four genomic locations for further analysis. Genomic DNA was extracted from tissue samples, followed by bisulfite conversion, locus-specific polymerase chain reaction (PCR) amplification, and pyrosequencing. Results: Four loci—cg-9652652, cg-11536474, cg-3867465, and cg-10122865—along with several adjacent CpG sites, were found to be hypermethylated in buccal samples compared to other tissue types. The difference in DNA methylation of buccal samples was statistically significant (p < 0.0001) compared to other tissues, indicating the potential usefulness of these loci for forensic tissue identification. Two additional studies were conducted: (a) a species specificity study and (b) a mixture study involving two different tissue types. The species specificity study showed that the primers used in the assay were specific to primates and humans. They did not amplify five non-primate samples, while the two primate samples—chimpanzee and rhesus—provided usable methylation data. The mixture study involved DNA from two different tissues—buccal samples and semen—combined in varying proportions. The results showed a decrease in the overall percentage of DNA methylation at the locus cg-9652652 as well as five adjacent CpG sites when the amount of buccal cell DNA in the mixture was reduced. Conclusion: The specificity of the primers and the significant differences in percent DNA methylation between buccal cells and other tissues make these markers excellent candidates for forensic tissue identification. Full article

Giardia duodenalis is a flagellated protozoan pathogen causing parasitic enteric disease outbreaks worldwide. Among detection methods, loop-mediated isothermal amplification (LAMP) has high selectivity and sensitivity, and the detection time is lower than that of conventional molecular methods. In this study, three published Giardia LAMP primer sets were tested and adapted to touchdown LAMP conditions. The measurement time, the volume of reagents, the effect of the denaturation step, different kinds of polymerases, and the presence or absence of betaine on the reaction were tested and evaluated. Based on the results of this study, the 66–60 °C range touchdown LAMP with the use of betaine, 90 °C denaturation step, Bst 2.0 WarmStart^® DNA Polymerase, and the primer set of Momoda et al. were the optimal conditions. We increased the analytical sensitivity of the LAMP reaction to 7.8- and 8-fold higher than the previously published methods for G. duodenalis assemblages A and B, with detection limits of 20 and 19.5 fg/assay, respectively, instead of 156 fg/assay. The detection time was less than 49 min for G. duodenalis assemblage A and less than 35 min for assemblage B, compared to the previously published 60 min. Our optimized LAMP protocol can be directly applied to improve Giardia LAMP tests in routine testing laboratories, could be implemented in standard diagnostic or environmental monitoring workflows, and can be used for the development of Giardia LAMP point-of-care devices or high-throughput systems. Full article

Diabetic kidney disease (DKD) displays a high prevalence of cardiovascular and cerebrovascular disease. Both the kidney and the brain share common pathogenic mechanisms, such as inflammation, endothelial dysfunction, oxidative stress, and mitochondrial dysfunction. The aim of this study was to establish a potential association of cerebral vessel remodeling and its related functional impairment with biomarkers of inflammation, oxidative stress, and mitochondrial dysfunction in the early stages of DKD in type 2 diabetes mellitus (DM) patients. A cohort of 184 patients and 39 healthy controls was assessed concerning serum and urinary stromal cell-derived factor-1 (SDF-1), P-selectin, advanced oxidation protein products (AOPPs), urinary synaptopodin, podocalyxin, kidney injury molecule-1 (KIM-1), and N-acetyl-β-(D)-glucosaminidase (NAG). The quantification of the mitochondrial DNA copy number (mtDNA-CN) and nuclear DNA (nDNA) in urine and peripheral blood was conducted using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Using TaqMan tests, the beta-2 microglobulin nuclear gene (B2M) and the cytochrome b (CYTB) gene, which encodes subunit 2 of NADH dehydrogenase (ND2), were evaluated. The MtDNA-CN is the ratio of mitochondrial DNA to nuclear DNA copies, ascertained through the examination of the CYTB/B2M and ND2/B2M ratios. The intima-media thickness (IMT) measurements of the common carotid arteries (CCAs), along with the pulsatility index (PI) and resistivity index (RI) of the internal carotid arteries (ICAs) and middle cerebral arteries (MCAs), were obtained through cerebral Doppler ultrasonography (US). Additionally, the breath-holding index (BHI) was also measured by cerebral Doppler US. PI-ICAs, PI-MCAs, CCAs-IMT, RI-MCAs, and RI-ICAs demonstrated direct relationships with SDF-1, P-selectin, AOPPs, urine mtDNA, podocalyxin, synaptopodin, NAG, and KIM-1 while showing indirect correlations with serum mtDNA and the eGFR. In contrast, the BHI had negative correlations with SDF-1, P-selectin, AOPPs, urine mtDNA, synaptopodin, podocalyxin, KIM-1, and NAG while showing direct associations with serum mtDNA and the eGFR. In conclusion, a causative association exists among SDF-1, P-selectin, and AOPPs, as well as mitochondrial dysfunction, in early diabetic kidney disease (DKD) and significant cerebrovascular alterations in patients with type 2 diabetes mellitus and normoalbuminuric DKD, with no neurological symptoms. Full article

Hepatitis B is a serious infectious disease that threatens the health of all mankind. In this study, we isolated and extracted hydroxytyrosol from Lindernia ruellioides with anti-hepatitis B virus (HBV) activity. The structure of hydroxytyrosol was identified by the nuclear magnetic resonance technique. HepG2.2.15 cell models were used to detect the anti-HBV activity and liver protection of hydroxytyrosol in vitro. Hydroxytyrosol can inhibit hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg). The IC₅₀ values of HBsAg and HBeAg were 4.02 mg/L and 5.19 mg/L, respectively. At the highest concentration of hydroxytyrosol, the inhibition rates of supernatant and intracellular HBV DNA were 75.99% and 66.33%, respectively. Hydroxytyrosol was less toxic to normal human hepatocytes. Molecular docking showed that hydroxytyrosol was bound to three amino acid residues of HBV polymerase with a binding energy of −7.0 kcal/mol. This study provided data for the development and utilization of Lindernia ruellioides and the research and development of anti-hepatitis B virus drugs. Full article

Rickettsiae are Gram-negative and obligate intracellular bacteria that cause rickettsioses. These pathogens are typically transmitted by arthropod vectors, such as ticks, mesostigmatid mites, and fleas. Rickettsiae are responsible for many emerging infectious diseases worldwide and are the second most frequently reported cause of non-malarial febrile illnesses in Southeast Asia. However, in Malaysia, studies on the prevalence and distribution of rickettsiae have primarily focused on humans, with limited data on these bacteria in vectors and small mammal hosts. Thus, this study aims to investigate the presence of Rickettsia spp. in small mammals and their associated mesostigmatid mites collected from potential rickettsioses areas in Selangor. Animal trapping was conducted across three different ecological study sites comprising a recreational area, agricultural land, and coastal area. A total of 41 small mammals and 363 mesostigmatid mites were collected and identified. Ten percent of the total individual mites were processed for morphological examination, and the remaining mites were then pooled by hosts, with five individual mites per tube, for DNA extraction. The collected samples, comprising blood, animal tissue, and pooled mites, were subjected to DNA extraction and were screened for Rickettsia spp. via nested polymerase chain reaction (PCR), targeting the citrate synthase-encoding gene (gltA) and outer membrane protein B gene (ompB). Interestingly, two pools (3.33%) of Laelaps spp. recovered from Maxomys whiteheadi and Bandicota indica, collected from a recreational area, tested positive for Rickettsia spp. Sequence analysis and phylogenetic tree of the ompB gene revealed the presence of Rickettsia felis in both laelapid mite pools. To our knowledge, this study provides the first molecular detection of R. felis in Laelaps spp. in Malaysia. Full article

Uncoupling protein 2 (UCP2) plays an important role in normal cells because it mitigates the cytotoxic effect of reactive oxygen species (ROS). However, its overexpression in cancer cells is related to drug resistance and increased cell proliferation due to a decrease in ROS production. In this context, molecules that regulate or block UCP2 have potential as anticancer agents. (-)-Epicatechin, a flavonoid that inhibits cell proliferation, increases ROS, and induces apoptosis in cancerous cells, was evaluated for its effects on UCP2 gene expression. For this purpose, the real-time quantitative polymerase chain reaction (qRT–PCR) and Western blotting were performed in MDA-MB-231 and MCF-10A cells to determine the effects of (-)-epicatechin on UCP2 expression. Furthermore, the impact of (-)-epicatechin on cell viability was also determined. To analyze the transcriptional regulation of the UCP2 gene by (-)-epicatechin, a 5′-region of the human UCP2 gene (−2093/+297) was amplified, sequenced, cloned, and inserted into a reporter plasmid. To analyze the promoter activity and regulatory motif involved in the effects of (-)-epicatechin, several deletions of the UCP2 promoter were generated and transfected into MDA-MB-231 and MCF-10A cells. An electrophoretic mobility shift assay (EMSA) was carried out to detect the interaction between DNA and proteins involved in the effect of (-)-epicatechin. The increased expression of the UCP2 gene in MDA-MB-231 cells was decreased by (-)-epicatechin, and the opposite effect was observed in MCF-10A cells. The promoter region of the human UCP2 gene (−2093/+297) showed activity, which was decreased by (-)-epicatechin. A sequence of 117 bp located at position −109 b to +8 b has a fragment of 90 bp that is related to the (-)-epicatechin effect. Bioinformatics analysis and EMSA of this sequence revealed the presence of a regulatory site for a protein with zinc fingers. The presence of a response element to (-)-epicatechin in the human UCP2 promoter revealed that the inhibition of this gene in MDA-MB-231 breast cancer cells occurred at the transcriptional level. In this study, we propose the mechanism of action of (-)-epicatechin that could aid in cancer treatment. Full article

Background: The World Health Organization has indicated that airport noise is strongly associated with cardiovascular disease, with vascular inflammation identified as the primary mechanism. Therefore, long-term exposure to airport noise is considered far more harmful than other types of noise. However, there remains a lack of research into the mechanisms underlying long-term exposure to airport noise and harm to the human body. Methods: A mouse model was established and exposed to airport noise at a maximum sound pressure level of 95 dB(A) and an equivalent continuous sound pressure level of 72 dB(A) for 12 h per day over a period of 100 days. Quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expression levels of pro-inflammatory and anti-inflammatory factors. Enzyme-linked immunosorbent assay (ELISA) was used to detect LPS, LTA, TMA, and TMAO levels. Intestinal flora composition was analyzed by 16S rDNA sequencing, and targeted metabolomics was employed to determine the levels of serum short-chain fatty acids. Results: Long-term airport noise exposure significantly increased systolic blood pressure, diastolic blood pressure, and mean blood pressure (p < 0.05); significantly increased the mRNA expression levels of oxidative stress parameters (nuclear matrix protein 2, 3-nitrotyrosine, and monocyte chemoattractant protein-1) (p < 0.05); significantly increased pro-inflammatory factors (interleukin 6 and tumor necrosis factor alpha) (p < 0.05); significantly decreased the mRNA expression level of anti-inflammatory factor interleukin 10 (p < 0.05); and significantly increased the content of LPS and LTA (p < 0.05). The composition of the main flora in the intestinal tract was structurally disordered, and there were significant differences between the noise-exposed and control groups at the levels of the phylum, family, and genus of bacteria. β-diversity of the principal component analysis diagrams was clearly distinguished. Compared with those of the control group, TMA-producing bacteria and levels of TMA and TMAO were significantly reduced, and the serum ethanoic acid and propanoic acid levels of the noise-exposed group were significantly decreased (p < 0.05). Conclusions: Long-term airport noise exposure causes significant elevation of blood pressure and structural disruption in the composition of the intestinal flora in mice, leading to elevated levels of oxidative stress and inflammation, resulting in metabolic disorders that lead to significant changes in the production of metabolites. Full article