Polymerase Chain Research Articles

Diagnostic potential of salivary IL-1β, IL-8, SAT, S100P, and OAZ1 in oral squamous cell carcinoma, oral submucous fibrosis, and oral lichen planus based on findings from a Sri Lankan cohort

The research examined the salivary concentrations of various biomarkers, such as OAZ1, SAT, S100P, IL-1β, and IL-8 aiming to detect early-stage oral squamous cell carcinoma (OSCC). These biomarkers show potential as indicators for detecting both pre-cancerous and cancerous states within the oral cavity. Analyzing these specific molecules in saliva could help clinicians enhance diagnostic accuracy and refine early detection methods for OSCC. The research encompassed a cohort of nine OSCC patients, ten with oral submucous fibrosis (OSF), eleven individuals with oral lichen planus (OLP), and ten healthy controls. The study focused on assessing the expression levels of key biomarkers—IL-1β, IL-8, SAT, S100P, and OAZ1 mRNA—in extracellular RNA extracted from saliva samples. This evaluation was conducted using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) with sequence-specific primers. Additionally, receiver operating characteristic curve (ROC) curve analysis was employed to gauge the efficacy of these biomarkers in detecting OSCC. Based on the results we observe, when these five biomarkers are used together, they give a 90% predictive probability for patients with OLP, an 80% predictive probability for OSF, and an impressive 100% predictive probability for patients with OSCC (AUC = 1.000, p = 0.000). This study demonstrates the efficacy of salivary transcriptome diagnostics in detecting OSCC. This novel clinical technique has the potential to be a powerful, efficient, and reliable tool for early detection of cancer. Salivary transcriptomes can be further analyzed to evaluate their effectiveness in other important illness contexts and for regular health monitoring.

Urinary sediment mRNA as a potent biomarker of IgA nephropathy

BackgroundThe quantification of mRNA expression in urinary sediments is a reliable biomarker for various diseases. However, few studies have investigated the clinical relevance of urinary mRNA levels in IgA nephropathy (IgAN). Thus, we investigated the expression of urinary mRNAs and their clinical significance in IgAN.MethodsOverall, 200 patients with biopsy-proven IgAN, 48 disease controls, and 76 healthy controls were enrolled. We identified the differential expression of mRNAs in renal tissue between patients with IgAN and normal subjects using the Gene Expression Omnibus dataset and selected candidate mRNAs. mRNA expression in the urinary sediment was measured using quantitative real-time polymerase chain reaction. Associations between urinary mRNA levels and clinicopathological parameters were analyzed and the predictive value of mRNAs for disease progression was evaluated.ResultsThe urinary expression of CCL2, CD14, DNMT1, FKBP5, Nephrin, and IL-6 was significantly upregulated in patients with IgAN compared with healthy controls. C3, FLOT1, and Podocin levels were significantly correlated with renal function, where C3, FLOT1, and TfR levels were significantly correlated with urinary protein excretion. During follow-up, 26 (13.0%) patients with IgAN experienced disease progression, defined as a greater than 50% reduction in the estimated glomerular filtration rate or progression to end-stage renal disease. Urinary mRNA levels of FLOT1 (HR 3.706, 95% CI 1.373–10.005, P = 0.010) were independently associated with an increased risk of disease progression.ConclusionsOur results suggest that urinary sediment mRNAs are a useful biomarker in IgAN patients. Further studies with larger sample sizes and longer follow-up durations are required.

Integrating Clinical and Microbiological Expertise to Improve Vaginal Candidiasis Management.

Vaginal candidiasis (VC) is a prevalent condition among women of reproductive age and poses a significant global public health challenge. However, the disease is often diagnosed and treated without mycological information. We investigated the epidemiology, laboratory diagnostics, and antifungal susceptibility of VC. We included 300 women from Çukurova University Obstetrics and Gynecology outpatient clinic in Adana, Türkiye. Participants underwent a health survey and provided vaginal swab samples for microscopic examination and fungal culture. The microscopic analysis involved wet-mount and gram-stained slides, whereas fungal identification involved CHROMAgar Candida, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and real-time polymerase chain reaction high-resolution melting analysis (RT-PCR HRMA). Antifungal susceptibility tests were conducted at pH 7 and 4 using the CLSI document M44-A2. Of the 106 women with positive fungal cultures, 86.8% were diagnosed with VC, whereas 13.2% showed Candida colonization. Among those with VC, 55.4% had acute and 44.6% had recurrent VC; a family history of allergies increased the risk for both types. We recovered 115 yeast isolates, predominantly C. albicans, C. glabrata, and C. krusei. Diagnostic accuracy of CHROMAgar Candida was 91.3% for the most common isolates, and HRMA was consistent in differential diagnosis. Antifungal resistance varied with pH; susceptibility to fluconazole, itraconazole, and ketoconazole decreased at pH 4, whereas susceptibility to miconazole increased. Our findings underscore the need for a diagnostic algorithm and enhanced collaboration between clinicians and microbiologists to improve VC management. Recommendations include using Gram staining, CHROMAgar Candida, MALDI-TOF MS, and antifungal susceptibility tests at both pH levels.

Surface-functionalized UIO-66-NH2 for dual-drug delivery of vancomycin and amikacin against vancomycin-resistant Staphylococcus aureus

BackgroundConventional antibacterial compounds can inhibit the growth of microorganisms, but their adverse effects and the development of drug limit their widespread use. The current study aimed to synthesize PEG-coated UIO-66-NH2 nanoparticles loaded with vancomycin and amikacin (VAN/AMK-UIO-66-NH2@PEG) and evaluate their antibacterial and anti-biofilm activities against vancomycin-resistant Staphylococcus aureus (VRSA) clinical isolates.MethodsThe VAN/AMK-UIO-66-NH2@PEG were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) to determine their size, polydispersity index (PDI), encapsulation efficiency (EE%), zeta-potential, drug release profile, and physical stability. Antibacterial activity was evaluated using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays. Biofilm formation by VRSA was assessed using the crystal violet (CV) and minimum biofilm eradication concentration (MBEC) assays. The effect of sub-MIC concentrations of the formulations on the expression of biofilm-related genes (icaA, icaD) and resistance-related genes (mecA, vanA) was investigated using quantitative real-time polymerase chain reaction (RT-qPCR).ResultsAs demonstrated by MIC, MBC and time-kill assay, the VAN/AMK-UIO-66-NH2@PEG nanoparticles exhibited enhanced antibacterial activity against VRSA isolates compared to free drugs and prepared formulations. Furthermore, CV and MBEC tests indicated that the VAN/AMK-UIO-66@NH2/PEG can reduce biofilm formation dramatically compared to VAN/AMK and VAN/AMK-UIO-66@NH2, due to its great drug release properties. This study also found that the expression level of the mecA, vanA, icaA, and icaD genes in VAN/AMK-UIO-66@NH2/PEG treated VRSA isolates was substantially decreased compared to other groups.ConclusionsThese findings highlighted the efficiency of VAN/AMK-UIO-66@NH2/PEG in combating antimicrobial resistance and biofilm formation in VRSA isolates. Future studies, particularly in vivo models, are necessary to evaluate the safety, efficacy, and clinical applicability of these nanoparticles for the treatment of bacterial infections.

CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus.

Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mecA, a 5.5-fold decrease in grlA, a 6-fold decrease in grlB, and a 4-fold decrease in aacA expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grlB and aacA genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.

PNSC5325 prevents acute respiratory distress syndrome by alleviating inflammation and inhibiting extracellular matrix degradation of alveolar macrophages

BackgroundAcute respiratory distress syndrome (ARDS) is characterized by severe inflammation and significant extracellular matrix (ECM) degradation in the lungs. Our prior research identified the CtBP2-p300-NF-κB (C-terminal-binding protein 2-histone acetyltransferase p300-nuclear factor kappa B) transcriptional complex as critical in ARDS by activating pro-inflammatory cytokine genes. MethodsAn ARDS mouse model was established using intratracheal instillation of lipopolysaccharide (LPS). Small molecules that inhibit the CtBP2-p300 interaction were identified through AlphaScreen. RNA sequencing (RNA-Seq) was conducted to determine differential gene expression. Immunoprecipitation and co-immunoprecipitation analyzed protein interactions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunoblotting detected gene and protein expression. Histological staining evaluated tissue damage. ResultsThrough AlphaScreen, two natural compounds, PNSC2477 and PNSC5325, were identified for their ability to inhibit the CtBP2-p300 interaction. While PNSC2477 demonstrated toxicity and was deemed unsuitable for further research, PNSC5325 exhibited minimal toxicity. PNSC5325 effectively inhibited the CtBP2-p300 interaction and reduced pro-inflammatory cytokine gene expression. RNA-Seq analysis of PNSC5325-treated cells indicated significant suppression of pro-inflammatory cytokine genes and matrix metalloproteinases (MMPs). Further molecular studies revealed that the CtBP2-p300 complex, in conjunction with activator protein 1 (AP1), activates MMP expression. PNSC5325 simultaneously suppressed both pro-inflammatory cytokines and MMPs by targeting the CtBP2-p300 complex. In LPS-injected mice, PNSC5325 administration significantly reduced ARDS incidence by inhibiting inflammatory and MMP genes. ConclusionThese findings suggest that PNSC5325 protects against ARDS by inhibiting key inflammatory and ECM degradation pathways, highlighting its potential as a novel therapeutic agent for ARDS and paving the way for further clinical investigations.

Sensitive Detection of Gynecological Cancer Recurrence Using Circulating Tumor DNA and Digital PCR: A Comparative Study with Serum Biochemical Markers

Early detection of recurrences in gynecological cancers is crucial for women’s health. Circulating tumor DNA (ctDNA) analysis through liquid biopsy offers a promising approach for monitoring disease progression and identifying relapses. This study investigated the utility of digital Polymerase Chain Reaction (dPCR) for ctDNA detection in three gynecological cancer patients with clinically confirmed relapses during a two-year post-surgical follow-up. Patient-specific tumor mutations were identified through whole-exome sequencing (WES) and confirmed via Sanger sequencing. dPCR probes targeting these mutations were used to quantify the ctDNA levels in plasma samples collected throughout the follow-up period, and the findings were compared with standard serum biochemical markers. In two patients, persistent positive dPCR signals for the selected mutations were detected after tumor removal, with ctDNA levels progressively increasing even after post-surgical chemotherapy. Notably, dPCR identified elevated ctDNA levels before an increase in the cancer antigen 125 (CA125) biochemical marker was observed. In the third patient, no ctDNA signals from the two selected mutations were detected despite clinical evidence of recurrence, suggesting the emergence of new mutations. While this study highlights the promise of dPCR for early recurrence detection in gynecological cancers, it also underscores the critical need for comprehensive mutation panels to overcome the inherent challenges posed by tumor heterogeneity and the emergence of new mutations during disease progression.

Determination and analysis of gene expression involved in glucose metabolism under the conditions of the development of experimental diabetes of dexamethasone type (type 2 diabetes)

Type 2 diabetes is one of the most common and serious chronic diseases today, which has become a global health care problem. Type 2 diabetes accounts for about 90–95 % of all cases of diabetes and significantly affects patients’ quality life due to the development of numerous complications, such as cardiovascular diseases, chronic renal failure, retinopathy, neuropathy. Modern research in the field of diabetology pays considerable attention to the understanding of the genetic mechanisms of the pathogenesis of type 2 diabetes, which makes it possible to develop new approaches to its diagnosis and treatment. Individualization of laboratory diagnostics and treatment, which considers the genetic, metabolic and clinical characteristics of each patient, is a key direction in improving the effectiveness of the treatment of type 2 diabetes. Type 2 diabetes is a complex polygenic disease, that develops under the influence of both genetic and external factors, which requires an integrated approach to its diagnosis, treatment and prevention. Taking into account the constant increase in the prevalence of this disease, the relevance of scientific research in this area is beyond doubt. The development of new pharmacological agents, improvement of laboratory diagnostic strategies and individualization of treatment are key directions for overcoming the problem of type 2 diabetes and improving the quality of life of patients. The aim of the work: identification and analysis of genes panel, involved in glucose metabolism under the conditions of the development of experimental type 2 diabetes. Materials and methods. Analysis of the gene expression, involved in glucose metabolism was performed using the real-time reverse transcription polymerase chain reaction method CFX-96 Touch™ (Bio-Rad, USA) using the RT2Profiler™ PCR Array Rat Diabetes kit (QIAGEN, Germany). Results. Based on the results of the PCR study, the activity of the studied genes involved in glucose metabolism can be divided as follows: G6pc, Gpd1 – genes with high expression compared to the control group of animals; Ace, Acly, Foxg1, Foxp3, Gcgr, Gck, Gsk3b, Hmox1, Pygl, Snap23, Snap25 – genes with low expression compared to the control group of animals; Cebpa, Dpp4, Sell – genes in which no changes were detected in the samples in relation to the control group of animals; Ccr2, Fbp1, Gcg – genes, whose expression was not detected. Conclusions. The development of dexamethasone type 2 diabetes significantly (where ∆∆Ct <30) increases the expression of Gpd1 genes by 8 times and G6pc by 2 times compared to the control group of animals. During the development of type 2 dexamethasone diabetes, significantly (where ∆∆Ct <30) the Gsk3b and Hmox1 genes had a 17-fold low expression; Pygl at 11; Foxg1 in 7; Gck in 6; Ace and Foxp3 in 4; Acly in 3; Gcgr, Snap23, Snap25 in 2 times compared to the control group of animals. The expression of Ccr2, Fbp1, Gcg genes during the development of type 2 dexamethasone diabetes was not detected.

SNAP25 as a prognostic marker in transcriptome analysis of meningioma.

Meningiomas are the most common intracranial tumors and their diagnosis relies mostly on neuroimaging and histology. However, the histology grades cannot predict the outcome exactly and some meningiomas tend to recur after resection of even benign tumors. Therefore, it is necessary to explore prognostic and diagnostic molecular targets. Differential expression analysis between meningiomas and meninges was performed based on the merged data of GSE43290 and GSE84263. Next, we performed gene set enrichment analysis (GSEA), immune cell infiltration analysis, protein-protein interaction analysis, and survival analysis using public data. The expression level of Synaptosome-associated-protein-25kDa (SNAP25) was verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting in meningioma tissues. There were 263 upregulated and 592 downregulated genes identified in meningiomas by differential expression analysis. GSEA results revealed that meningiomas were negatively related to the pathway of soluble N-ethylmaleimide sensitive factor attachment protein receptor interactions in vascular transport and chemokine signaling. SNAP25 was characterized as a hub gene and downregulated in meningiomas. The Kaplan-Meier plot indicated that high expression of SNAP25 is a favorable factor. SNAP25 was downregulated and identified as a potential prognostic marker in meningioma.

Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques

Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods adopting the Jonstrup assay (J assay). Chimeric plasmid DNA (cpDNA) harboring various pathogen genes and the target site of the J assay was constructed. Our findings revealed that the J assay could detect a single copy of the target gene similar to digital droplet PCR. The detection sensitivity of each established real-time PCR method for various disease diagnoses was evaluated using the cpDNA. Although most methods showed high sensitivity, similar to that of the J assay, the VHS Garver and SARS-CoV-2 diagnostic methods exhibited detection sensitivities tenfold lower than that of the J assay. To ensure accuracy of results and avoid genetic contamination from positive controls, we introduced an additional probe attachment site emitting distinct fluorescent signals within the cpDNA. Target genes and exogenous sequences within the plasmid DNA were simultaneously detected in a single assay using this unique method. Our approach will help improve the sensitivity of diagnostic methods and develop novel diagnostic methods based on molecular techniques.

Circ_0068655 silencing ameliorates hypoxia-induced human cardiomyocyte injury by regulating apoptotic and inflammatory responses.

There is growing evidence suggesting that the dysregulation of circular RNAs (circRNAs) plays a significant role in various myocardial disorders, including myocardial ischemia (MI). This study aimed to explore the function of hsa_circ_0068655 (circ_0068655) in hypoxia-induced cardiomyocyte injury. Human AC16 cardiomyocyte cells were cultured under anaerobic condition to induce an in vitro model of MI. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate staining and caspase-3 and caspase-9 activity assays. Cell proliferation was analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay. Inflammation was evaluated by enzyme-linked immunosorbent assays. Circ_0068655, miR-370-3p and BCL-2-like 11 (BCL2L11) expression were detected by real-time quantitative polymerase chain reaction or western blotting. The target interactions among circ_0068655, miR-370-3p and BCL2L11 were predicted using bioinformatics tools and validated using dual-luciferase reporter assays and RNA immunoprecipitation assays. Hypoxia treatment led to upregulated expression of circ_0068655 and BCL2L11, and downregulated expression of miR-370-3p in AC16 cells. This treatment also resulted in reduced cell viability, increased apoptosis rate, elevated caspase-9/3 activities and cleavage, and enhanced production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Notably, knockdown of circ_0068655 alleviated these detrimental effects. In addition, circ_0068655 silencing-mediated effects were restored by decreasing miR-370-3p expression in hypoxia-treated AC16 cells. Moreover, ectopic BCL2L11 expression remitted the effects of miR-370-3p overexpression on hypoxia-treated AC16 cells. Mechanistically, circ_0068655 was found to act as a sponge for miR-370-3p, thereby regulating BCL2L11 expression. Circ_0068655 silencing ameliorated hypoxia-induced human cardiomyocyte injury through the miR-370-3p/BCL2L11 axis.

Designing and Validating a Survey for National-Level Data During the COVID-19 Pandemic in Sri Lanka: Cross-Sectional Mobile Phone Surveys.

The COVID-19 pandemic has generated a demand for timely data, resulting in a surge of mobile phone surveys for tracking the impacts of and responses to the pandemic. Mobile phone surveys have become a preferred mode of data collection across low- and middle-income countries. This study piloted 2 population-based, cross-sectional mobile phone surveys among Sri Lankan residents in 2020 and 2021 during the COVID-19 pandemic. The surveys aimed to gather data on knowledge, attitudes, and practices, vaccine acceptability, availability, and barriers to COVID-19 testing, and use of a medicine distribution service. The study used Surveda, an open-source survey tool developed by the NCD (noncommunicable disease) Mobile Phone Survey Data 4 Health Initiative, for data collection and management. The surveys were conducted through interactive voice response using automated, prerecorded messages in Sinhala, Tamil, and English. The sample design involved random sampling of mobile phone numbers, stratified by sex, proportional to the general population. Eligibility criteria varied between surveys, targeting adults aged 35 years and older with any noncommunicable disease for the first survey and all adults for the second survey. The data were adjusted to population estimates, and statistical analysis was conducted using SAS (SAS Institute) and R software (R Core Team). Descriptive statistics, Rao-Scott chi-square tests, and z tests were used to analyze the data. Response rates, cooperation rates, and productivity of the sampling approach were calculated. In the first survey, n=5001, the overall response rate was 7.5%, with a completion rate of 85.6%. In the second survey, n=1250, the overall response rate was 10.9%, with a completion rate of 61.9%. Approximately 3 out of 4 adults reported that they avoided public places (888/1175, 75.6%), more than two-thirds avoided public transportation (808/1173, 68.9%), and 9 out of 10 practiced physical distancing (1046/1167, 89.7%). Approximately 1 out of 10 Sri Lankan persons reported being tested for COVID-19, and the majority of those received a polymerase chain reaction test (112/161, 70%). Significantly more males than females reported being tested for COVID-19 (98/554, 17.8% vs 61/578, 10.6%, respectively; P<.001). Finally, the majority of adult Sri Lankan people reported that they definitely or probably would get the COVID-19 vaccination (781/1190, 65.7%). The surveys revealed that, overall, the adult Sri Lankan population adhered to COVID-19 mitigation strategies. These findings underscore the use of mobile phone surveys in swiftly and easily providing essential data to inform a country's response during the COVID-19 pandemic, obviating the need for face-to-face data collection.

Ameliorating Immune-dependent Inflammation and Apoptosis by Targeting TLR4/MYD88/NF-ᵰ5B Pathway by Celastrol Mitigates the Diabetic Reproductive Dysfunction.

This study aimed to examine the protective effect of celastrol on testicular dysfunction in diabetic rats and the potential underlying mechanisms. All rats included in the study were divided into four groups: a control group treated with sodium citrate buffer and vehicle), a celastrol-treated control group, a streptozotocin (STZ)-induced diabetic group following insulin resistance, and a celastrol-treated diabetic group. Serum glucose, triglyceride, total cholesterol, high-density lipoprotein cholesterol, interleukin (IL)-1β, tumor necrosis factor-alpha, and testosterone levels were measured. In addition, the levels of testicular homogenate superoxide dismutase and malondialdehyde were assessed. Furthermore, testicular tissue relative TLR4, NF-ᵰ5B, and MYD88 expression were quantitatively measured using polymerase chain reaction. Histopathological and immunohistochemical studies were also conducted. The results revealed that treatment with celastrol significantly reduced TLR4, MyD88, NF-ᵰ5B expressions and the levels of inflammatory mediators such as tumor necrosis factor-alpha and IL-1ᵯD in the testicular tissue of treated rats. These findings suggest has the potential to be effective in the treatment of diabetes-induced testicular injury by inhibiting testicular inflammation, apoptosis, and oxidative stress.

Self-testing for human papillomavirus as a screening method for identifying risk groups

Due to the high prevalence of cervical cancer, it is necessary to take measures aimed at reducing its frequency through vaccination, screening, detection and treatment of precancerous conditions. The article presents a systematic analysis of scientific data on the use of testing for self-sampling for human papillomavirus as a tool to improve the effectiveness of screening and detection of precancerous diseases of the cervix. It has been shown that the use of such tests, which are based on polymerase chain reaction (PCR), have similar clinical accuracy compared to sampling by a doctor, and thus this option is promising for expanding the coverage of cervical cancer screening. The presence of expected economic efficiency also determines the relevance and prospects for these tests, but further research is needed to assess their implementation in routine practice.

The GDF15 3′ UTR Polymorphism rs1054564 Is Associated with Diabetes and Subclinical Atherosclerosis

Growth differentiation factor 15 (GDF15) is a stress-response cytokine related to a wide variety of metabolic diseases. However, the impact of GDF15-specific genetic variants on the abovementioned conditions is poorly known. The aim of this study was to assess the impact of selected GDF15 single-nucleotide polymorphisms (SNPs) on metabolic disturbances and subclinical atherosclerosis. A cross-sectional study involving 153 participants of a metabolic patient-based cohort was performed. Three selected SNPs (rs888663, rs1054564 and rs1059369) in a locus on chromosome 19 including the GDF15 gene were genotyped by Polymerase Chain Reaction (PCR), and its relationship with the serum GDF15 levels, health status and clinical variables were analyzed. Of the three SNPs analyzed, only rs1054564 showed different distributions between the healthy volunteers and patients suffering lipid alterations and associated disorders. Accordingly, just the rs1054564 variant carriers showed a significant increase in GDF15 serum levels compared to the wild-type carriers. The group of variant carriers showed a higher frequency of individuals with diabetes, compared to the wild-type carrier group, without showing differences in other metabolic conditions. Additionally, the frequency of individuals with atherosclerotic carotid plaque was higher in the rs1054564 variant carriers than in the wild-type carriers. Logistic regression models identified that the presence of the rs1054564 variant carriers increase the likelihood for both diabetes and carotid plaque independently of confounding factors. Overall, the findings of this study identify the rs1054564 variant as a potential indicator for the likelihood of diabetes and subclinical atherosclerosis.

Design of Primers for the Detection of Parvovirus in Dogs and Cats Using the Same Polymerase Chain Reaction

Design of Primers for the Detection of Parvovirus in Dogs and Cats Using the Same Polymerase Chain Reaction

E-cadherin Single Nucleotide Variants Are Associated with Increasing Susceptibility to Periodontitis.

To investigate the association of E-cadherin single nucleotide polymorphisms (SNPs) with periodontitis and the potential of these SNPs for identifying susceptibility to periodontitis. Periodontal clinical parameters were recorded followed by collecting venous blood for DNA extraction. Polymerase chain reaction was used to amplify target segments of the E-cadherin gene. Determination of the genotype and allele frequencies was performed using Sanger sequencing. All statistical analyses were performed using GraphPad Prism (version 9) using a statistically significant difference of p < 0.05. A total of 207 participants were recruited into two groups of healthy controls (n = 105) and cases diagnosed with periodontitis stage 2 or 3, grade B or C (n = 102). Analyses indicated that the genotypes and alleles of rs3743674 and rs5030625 E-cadherin SNPs were significantly associated with periodontitis. Results from a binary regression model suggested that the presence of these SNPs may indicate susceptibility to periodontitis and increase the rate of progression. Linkage disequilibrium analysis indicated that E-cadherin variants rs3743674 and rs5030625, and rs10272115 and rs16260 were correlated in a nonrandom manner (r 2 = 0.638 and 0.495, respectively). E-cadherin gene variants, rs3743674 and rs5030625, were associated with the periodontitis phenotype. These biomarkers may identify individuals susceptible to periodontitis and the rate of disease progression.

First report of Trichoderma roseum causing tea leaf rot in China.

Tea (Camellia sinensis) is consumed worldwide for its numerous benefits and China lead the world production. In March 2023, leaf spots were observed on approximately 10% of tea plants in a 50-ha commercial tea plantation in Menghai (21°46'13"N, 100°30'6"E), Yunnan, China. Initial symptoms appeared as small spots, which progressively expanded and spread over the entire leaf surface. Subsequently, pale pink mold layers developed from the lesions (Fig. S1). To isolate the pathogen, small leaf pieces (3 × 3 mm) were cut from the margins of the lesions, sterilized with 75% ethanol for 30 sec and 0.5% NaClO for another 30 sec, and rinsed three times with sterile water. The pieces were placed on acidified potato dextrose agar (PDA) plates and incubated in darkness at 28°C. A total of 15 fungal isolates with identical morphologies were collected. The colonies appeared pale pink with white mycelia initially then turned orange-pink at the center and light white at the edges. After 10-15 days, exhibiting a powdery texture and concentric rings with uniform edges. Conidia were found at the apex peduncle and were inverted pear-shaped or oval, either non-septate (15.3 ± 2 × 7.8 ± 1.8 μm in size, n = 60) or septate, with a slightly constricted spore base featuring papillary projecvtions (14.8 ± 1.5 × 7.4 ± 0.7 μm in size, n = 60). The morphology closely resembled Trichoderma roseum (Oh et al. 2014). To confirm the species, the strain CYB5 was selected for identification by sequencing the ribosomal internal transcribed spacer (ITS) and large subunit (LSU) genes using polymerase chain reaction (PCR) (White et al.1990). The ITS (GenBank accession OR889657) and LSU (PQ270526) gene sequences exhibited 98% similarity with the Trichoderma roseum sequence KP317992 from NCBI database. A phylogenetic tree was constructed using MEGA 11 (Felsenstein 1981) based on the concatenated sequences (ITS and LSU) of the strain CYB5 and reference strains (Fig. S2). The analysis confirmed that CYB5 is T. roseum (Inácio et al. 2011). Pathogenicity tests were conducted on detached healthy tea leaves placed on wet filter paper in petri dishes. Micro-wounds were made on leaves using a sterilized needle, followed by inoculation with a 6-mm plug of CYB5. Control leaves were inoculates with fungus-free agar disks. The dishes were incubated at 25°C in the dark for 7 days. The leaves inoculated with CYB5 developed reddish brown to dark brown lesions around the inoculated sites, while control leaves remained asymptomatic. The fungus was reisolated from the lesion, and the isolates were morphologically identical to the original cultures. A second pathogenicity test was conducted on potted tea plants of the cultivar 'Yunkang No. 10.' Three plants scratched with a needle and three non-wounded plants were inoculated by spraying 20 ml of a spore suspension (105 spores/ml) of CYB5. Plants sprayed with sterile water served as controls. All plants were maintained in a growth chamber at 28°C, and 70% relative humidity. The lesions developed three days post-inoculation, and typical symptoms appeared after 10 days on spore-inoculated plants only. T. roseum was reisolated and reidentified based on the morphology and molecular analyses, thus fulfilling Koch's postulates. To our best knowledge, this is the first report of T. roseum causing tea leaf rot in China.

The impact of CTLA-4 gene polymorphism on the content of organ-specific antibodies in patients with autoimmune thyroiditis among the population of Azerbaijan

BACKGROUND: The risk of developing immune tolerance to self-antigens in autoimmune thyroiditis is largely associated with a mutation in the CTLA-4 gene, the product of which negatively regulates T-cell activity. AIM: To study the A49G (rs231775) CTLA-4 polymorphism in patients with autoimmune thyroiditis in a sample of Azerbaijani residents and the effect of the frequency of identified alleles and genotypes on the level of antibodies to thyroid peroxidase and thyroglobulin. MATERIAL AND METHODS: A study was conducted in 2021–2023 involving 170 patients with autoimmune thyroiditis and 65 individuals without thyroid pathology or other autoimmune diseases, who formed a comparison group. The groups were matched by gender and age. Based on the patient history, thyroid status study results, thyroid ultrasound data, and organ-specific antibody assessment, the patients were diagnosed with autoimmune thyroiditis. The routine thyroid status study results in the comparison group were within normal limits. Genotyping of the A49G (rs231775) polymorphism was performed using polymerase chain reaction followed by digestion of the reaction product with PspEI restriction endonuclease. The results of the quantitative studies were presented as the median (Me) and interquartile range (25th quartile; 75th quartile). The correspondence of genotype frequencies to the Hardy–Weinberg equilibrium was assessed using the χ2 criterion. RESULTS: The results revealed a statistically significant increase in the frequency of the G allele (48%) compared to the control group [33.8%; p=0.039, χ2=4.27, odds ratio (OR) 1.865, 95% confidence interval (CI) 1.028–3.382] and a decrease in the frequency of the A allele (51.2%) relative to the control group (66.1%; p=0.0389, χ2=4.27, OR=0.536, CI=0.296–0.973). In 22.4% of patients with the GG genotype (p=0.005, χ2=7.86, OR=0.237, 95% CI=0.088–0.635) and 55.6% of patients with the G allele (p=0.0012, χ2=10.43, OR=0.360, 95%, CI=0.192–0.674), thyroglobulin antibody titers were more than 100 IU/ml. Thyroid peroxidase antibody levels of 100 IU/ml or more were recorded in 22.7% of study participants with the GG genotype (p=0.030) and 50.0% with the G allele (p=0.048). CONCLUSION: Elevated titers of antibodies to thyroid peroxidase and thyroglobulin were detected in patients from the population of Azerbaijan with the G allele and homozygous genotype GG of the +49A/G polymorphism of the CTLA-4 gene.

Trem2/Tyrobp Signaling Protects Against Aortic Dissection and Rupture by Inhibiting Macrophage Activation in Mice.

The development of aortic dissection (AD) is closely associated with inflammation. The Trem2 (triggering receptor expressed on myeloid cells 2)/Tyrobp (TYRO protein tyrosine kinase-binding protein) signaling pathway critically regulates innate immunity and has emerged as an important target in cardiovascular diseases; however, its role in AD remains unclear. Transcriptome data from human and mouse ADs were used to perform differentially expressed gene-based protein-protein interaction network analyses. Tyrobp knockout (Tyrobp-/-), myeloid cell-specific Tyrobp-/- (Tyrobpfl/fl Lyz2cre), and Trem2 knockout (Trem2-/-) mice were given β-aminopropionitrile monofumarate in drinking water to induce AD. To dissect the role of macrophages in Tyrobp deficiency-mediated AD progression, macrophages were depleted using clodronate liposomes. Bulk and single-cell RNA sequencing, immunofluorescence staining, and quantitative real-time polymerase chain reaction were performed to assess inflammation and the underlying mechanisms of Tyrobp in AD. Network analysis identified Tyrobp as a hub gene of AD, with elevated levels observed in both human and mouse ADs. Global deletion and myeloid cell-specific deficiency of Tyrobp in mice significantly increased AD incidence and exacerbated extracellular matrix degradation and macrophage infiltration within the aortic wall. Macrophage depletion mitigated the adverse effects of Tyrobp deficiency on AD progression. Additionally, Tyrobp deficiency enhanced TLR (Toll-like receptor)-4 signaling and macrophage activation, which were abrogated by TLR4 inhibitors. Furthermore, deletion of the Tyrobp-associated receptor Trem2 significantly aggravated mouse AD development, whereas Trem2 agonist treatment conferred protection against AD. Our findings suggest a novel role for the Trem2/Tyrobp axis in AD development in mice. Enhancement of Trem2/Tyrobp signaling may represent a promising strategy for the prevention and treatment of AD. Future studies to clarify the role of Trem2/Tyrobp in human AD are warranted.