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Purpose: To examine the behavior and function of human gingival fibroblasts growing on healing abutments with or without laser-textured topography. Materials and Methods: Human primary gingival connective tissue fibroblasts were cultured on healing abutments with machined or laser-textured (Laser-Lok, BioHorizons) surfaces. Cellular and molecular responses were evaluated by a variety of tests, including cell density assay (WST-1), fluorescence microscopy, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and detachment tests. Results: The machined surface showed monodirectional traces and scratches from milling, whereas the laser-textured surface showed a distinct morphology consisting of monodirectional mesoscale channels (15-µm pitch) and woven oblique microridges formed within the channels. There were no differences in initial fibroblast attachment, subsequent fibroblast proliferation, or collagen production between the machined and laser-textured surfaces. Fibroblasts growing on a laser-textured surface were found to spread in one direction along the mesochannels, while cells growing on machined surfaces tended to spread randomly. Fibroblasts on laser-textured surfaces were 1.8 times more resistant to detachment than those on machined surfaces. An adhesive glycoprotein (fibronectin) and transmembrane adhesion linker gene (integrin ß-1) were upregulated on laser-textured surfaces. Conclusions: The increased fibroblast retention, uniform growth, and increased transcription of cell adhesion proteins compellingly explain the enhanced tissue-level response to laser-created and hybrid-textured titanium surfaces. These results provide a cellular and molecular rationale for the tissue reaction to this unique surface; in addition, they support its extended use, from implants and healing abutments to diverse prosthetic components where enhanced soft tissue responses would be desirable. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Viral lower respiratory tract infection (vLRTI) contributes to substantial morbidity and mortality in children. Diagnosis is typically confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) of nasopharyngeal specimens in hospitalized patients; however, it is unknown whether nasopharyngeal detection accurately reflects presence of virus in the lower respiratory tract (LRT). This study evaluates agreement between viral detection from nasopharyngeal specimens by RT-PCR compared with metagenomic next-generation RNA sequencing (RNA-Seq) from tracheal aspirates (TAs). DESIGN: This is an analysis of of a seven-center prospective cohort study. SETTING: Seven PICUs within academic childrens hospitals in the United States. PATIENTS: Critically ill children (from 1 mo to 18 yr) who required mechanical ventilation via endotracheal tube for greater than or equal to 72 hours. INTERVENTIONS: We evaluated agreement in viral detection between paired upper and LRT samples. Results of clinical nasopharyngeal RT-PCR were compared with TA RNA-Seq. Positive and negative predictive agreement and Cohens Kappa were used to assess agreement. MEASUREMENTS AND MAIN RESULTS: Of 295 subjects with paired testing available, 200 (68%) and 210 (71%) had positive viral testing by RT-PCR from nasopharyngeal and RNA-Seq from TA samples, respectively; 184 (62%) were positive by both nasopharyngeal RT-PCR and TA RNA-Seq for a virus, and 69 (23%) were negative by both methods. Nasopharyngeal RT-PCR detected the most abundant virus identified by RNA-Seq in 92.4% of subjects. Among the most frequent viruses detected, respiratory syncytial virus demonstrated the highest degree of concordance (κ = 0.89; 95% CI, 0.83-0.94), whereas rhinovirus/enterovirus demonstrated lower concordance (κ = 0.55; 95% CI, 0.44-0.66). Nasopharyngeal PCR was more likely to detect multiple viruses than TA RNA-Seq (54 [18.3%] vs 24 [8.1%], p ≤ 0.001). CONCLUSIONS: Viral nucleic acid detection in the upper versus LRT reveals good overall agreement, but concordance depends on the virus. Further studies are indicated to determine the utility of LRT sampling or the use of RNA-Seq to determine LRTI etiology.

Background: Coronavirus-19 disease (COVID-19) frequently causes coagulation disturbances. Data remains limited on the effects of microRNAs (miRNAs) on coagulation during COVID-19 infection. We aimed to analyze the comprehensive miRNA profile as well as coagulation markers and blood count in hospitalized COVID-19 patients. Methods: Citrated plasma samples from 40 patients (24 men and 16 women) hospitalized for COVID-19 were analyzed. Basic coagulation tests, von Willebrand factor (VWF), ADAMTS13, blood count, C-reactive protein, and 27 miRNAs known to associate with thrombosis or platelet activation were analyzed. MiRNAs were analyzed using quantitative reverse transcription polymerase chain reaction (RT qPCR), with 10 healthy controls serving as a comparator. Results: Among the patients, 15/36 (41%) had platelet count of over 360 × 109/L and 10/36 (28%) had low hemoglobin of < 100 g/L, while 26/37 (72%) had high VWF of over 200 IU/dL. Patients had higher levels of the miRNAs miR-27b-3p, miR-320a-3p, miR-320b-3p, and miR-424-5p, whereas levels of miR-103a-3p and miR-145-5p were lower than those in healthy controls. In total, 11 miRNAs were associated with platelet count. Let-7b-3p was associated with low hemoglobin levels of < 100 g/L. miR-24-3p, miR-27b-3p, miR-126-3p, miR-145-5p and miR-338-5p associated with high VWF. Conclusion: COVID-19 patients differentially express miRNAs with target genes involved in fibrinolysis inhibition, coagulation activity, and increased inflammatory response. These findings support the notion that COVID-19 widely affects hemostasis, including platelets, coagulation and fibrinolysis. [ABSTRACT FROM AUTHOR]

Objective: This study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation. Results: The rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations. [ABSTRACT FROM AUTHOR]

Background: Human parainfluenza virus-1 (HPIV-1) is a notable pathogen instigating acute respiratory tract infections in children. The article is to elucidate the epidemiological and genetic characteristics of HPIV-1 circulating in Hangzhou during the period of 2021–2022. Methods: A cohort of 2360 nasopharyngeal swabs were amassed and subsequently examined via RT-PCR, with HPIV-1 positive samples undergoing P gene sequencing. Results: The highest HPIV-1 infection rates were found in children aged between 3 and 6 years. A pronounced positive rate persisted through the latter half of 2021, with a notable decline observed in the initial half of 2022. All HPIV-1 strains could be clustered into 2 groups: Cluster 1, with strains similar to those found in Japan (LC764865, LC764864), and Cluster 2, with strains similar to the Beijing strain (MW575643). Conclusion: In conclusion, our study contributes to the comprehensive data on the epidemiological and genetic characteristics of HPIV-1 in pediatric patients from Hangzhou, post the COVID-19 peak. [ABSTRACT FROM AUTHOR]

Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2–4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15–30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed ƩS COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen's Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments. [ABSTRACT FROM AUTHOR]

In mammals, imprinted genes are characterised by a monoallelic expression, which is based on parental origin and is essential for both foetal and placental development. The ZFAT gene encodes a transcriptional factor, and its non‐coding antisense RNA, ZFAT‐AS1, overlaps with the ZFAT locus. Both ZFAT and ZFAT‐AS1 are maternally imprinted in human placentas. In bovines, the imprinting status of the ZFAT and ZFAT‐AS1 genes has yet to be reported. In this study, we analysed the allelic expression of three transcript variants (X1–X3) of the bovine ZFAT and ZFAT‐AS1 genes in somatic tissues and placentas using a single nucleotide polymorphism‐based method. The results showed that bovine ZFAT exhibited isoform‐specific paternal expression. The ZFAT X2 variant exhibited monoallelic expression in the bovine placentas and biallelic expression in the six bovine somatic tissues (heart, liver, spleen, lung, kidney and brain). However, the ZFAT X1 and X3 variants were biallelically expressed in both bovine tissues and placentas. A 311 bp bovine ZFAT‐AS1 complementary DNA (cDNA) sequence was obtained by aligning the human ZFAT‐AS1 cDNA sequence with the bovine genome and conducting reverse transcription polymerase chain reaction amplification. Bovine ZFAT‐AS1 have monoallelic expression in bovine placentas and somatic tissues. In addition, the DNA methylation of two regions was characterised, including the partial promoter, and exon 1 and intron 1 regions of ZFAT, and there were no differentially methylated regions. [ABSTRACT FROM AUTHOR]

Introduction Mass screening for SARS-CoV-2 using nasopharyngeal swabs (NPS) is costly, uncomfortable for patients, and increases the chance of virus exposure to health care workers. Therefore, this study focused on determining if self-collected unpreserved saliva can be an effective alternative to NPS collection in COVID-19 surveillance. Materials and Methods In this study, patients being tested for SARS-CoV-2 using NPS were asked to provide a saliva sample to compare their results. NPS samples were evaluated for SARS-CoV-2 using BioFire® FilmArray® Torch® or Cepheid® GeneXpert® systems while saliva samples were evaluated using an in-house developed reverse transcriptase polymerase chain reaction (RT-PCR) which targeted the Envelope (E) and Nucleocapsid (N) genes. Results Detection of SARS-CoV-2 using self-collected saliva was found to be only slightly less accurate (<5%) than testing using NPS. In addition, initial saliva RT-PCR identified 27 positive subjects, 18 of which provided amplicons sufficient for confirmatory sequencing. The sequencing data showed a genetic shift in the virus within our population sometime between 22 June and July 8, 2021 from Alpha to Delta variant. Conclusions The saliva sample collection method identifies the E gene in SARS COVID-2 samples which provides an alternative specimen source to the NPS. This identifies the S gene and ORF1ab. Saliva collection is more convenient to the patient, yields comparable results to NPS collection, and potentially increases Covid-19 surveillance. [ABSTRACT FROM AUTHOR]

Background: Focal segmental glomerulosclerosis (FSGS), a histologic pattern of injury in the glomerulus, is one of the leading glomerular causes of end-stage renal disease (ESRD) worldwide. Despite extensive research, the underlying biological alterations causing FSGS remain poorly understood. Studying variations in gene expression profiles offers a promising approach to gaining a comprehensive understanding of FSGS molecular pathogenicity and identifying key elements as potential therapeutic targets. This work is a meta-analysis of gene expression profiles from glomerular samples of FSGS patients. The main aims of this study are to establish a consensus list of differentially expressed genes in FSGS, validate these findings, understand the disease's pathogenicity, and identify novel therapeutic targets. Methods: After a thorough search in the GEO database and subsequent quality control assessments, seven gene expression datasets were selected for the meta-analysis: GSE47183 (GPL14663), GSE47183 (GPL11670), GSE99340, GSE108109, GSE121233, GSE129973, and GSE104948. The random effect size method was applied to identify differentially expressed genes (meta-DEGs), which were then used to construct a regulatory network (STRING, MiRTarBase, and TRRUST) and perform various pathway enrichment analyses. The expression levels of several meta-DEGs, specifically ADAMTS1, PF4, EGR1, and EGF, known as angiogenesis regulators, were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results: The identified 2,898 meta-DEGs, including 665 downregulated and 669 upregulated genes, were subjected to various analyses. A co-regulatory network comprising 2,859 DEGs, 2,688 microRNAs (miRNAs), and 374 transcription factors (TFs) was constructed, and the top molecules in the network were identified based on degree centrality. Part of the pathway enrichment analysis revealed significant disruption in the angiogenesis regulatory pathways in the FSGS kidney. The RT-qPCR results confirmed an imbalance in angiogenesis pathways by demonstrating the differential expression levels of ADAMTS1 and EGR1, two key angiogenesis regulators, in the FSGS condition. Conclusion: In addition to presenting a consensus list of differentially expressed genes in FSGS, this meta-analysis identified significant distortions in angiogenesis-related pathways and factors in the FSGS kidney. Targeting these factors may offer a viable strategy to impede the progression of FSGS. [ABSTRACT FROM AUTHOR]

Background: Inflammation and neuroinflammation are integral to the progression and severity of many diseases and are strongly associated with cardiovascular disease, cancer, autoimmune disorders, neurodegenerative disease, and neuropsychiatric disorders. These diseases can be difficult to treat without addressing the underlying inflammation, and, as such, a growing need has arisen for pharmaceutical treatments that target inflammatory mediators and signaling pathways. Our lab has investigated the therapeutic potential of the irreversible µ-opioid antagonist β-funaltrexamine (β-FNA) and discovered that acute treatment ameliorates inflammation in astrocytes in vitro and inhibits central and peripheral inflammation and reduces anxiety- and sickness-like behavior in male C57BL/6J mice. Now, our investigation has expanded to investigate the chronic pre-treatment effects of β-FNA on lipopolysaccharide (LPS)-induced inflammation and behavior in male C57BL/6J mice. Results: Micro-osmotic drug pumps were surgically inserted into the subcutaneous intrascapular space of male C57BL/6J mice. β-FNA or saline vehicle was continuously administered for seven days. On the sixth day, mice were given intraperitoneal injections of LPS or saline. An elevated plus maze test, followed by a forced swim test, were administered 24 h post-injection to measure sickness-, anxiety- and depressive-like behavior. Immediately after testing, frontal cortex, hippocampus, spleen, and plasma were collected. Levels of inflammatory chemokines C–C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 10 (CXCL10) were measured in tissues by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to assess expression of the enzyme indoleamine 2, 3-dioxygenase 1 (IDO1) and the NLR family pyrin domain-containing protein 3 (NRLP3) inflammasome in frontal cortex and spleen tissues. Chronic pre-treatment robustly decreased inflammation in the hippocampus, frontal cortex, and spleen and reduced or abolished anxiety- and sickness-like behavior (e.g., increased time spent motionless, increased time spent in a contracted position, and reduced distance moved). However, treatment with β-FNA alone increased both inflammation in the frontal cortex and anxiety-like behavior. Conclusion: These findings provide novel insights into the anti-inflammatory and behavior-modifying effects of chronic β-FNA pre-treatment and continue to support the therapeutic potential of β-FNA under inflammatory conditions. [ABSTRACT FROM AUTHOR]

Introduction: The study explored the effects of SHOX2 and RASSF1A DNA methylation in lung cancer (LC). Method: Bronchoalveolar lavage fluid (BALF) samples as well as LC and normal adjacent tissues were collected from 72 LC patients and 35 patients with benign pulmonary nodules. Quantitative analysis of SHOX2 and RASSF1A DNA methylation was performed in benign pulmonary nodules and different stages of LC. The diagnostic value of SHOX2 and RASSF1A DNA methylation in LC and benign pulmonary nodules was determined by receiver operating characteristics analysis. Gain/loss-of-function experiments were constructed in LC cells and mouse models of xenograft and pulmonary nodule metastasis. The levels of SHOX2 and transfer-associated genes were tested through quantitative reverse transcription polymerase chain reaction and Western blot. Malignant phenotype of LC cells was assessed by functional experiment. The tumor volume and weight of mice in xenograft models were measured. Pulmonary nodule metastasis was determined through HE staining assay. 5-azacytidine appeared as a positive control drug. Result: SHOX2 DNA methylation or RASSF1A DNA methylation had diagnostic efficiency in pulmonary nodules and early LC, with the two combined having better diagnostic value. SHOX2 expression was upregulated in LC. Similar to 5-azacytidine, SHOX2 knockdown inhibited LC cell viability, migration, and invasion in vitro as well as restrained LC tumorigenesis and pulmonary nodule metastasis in vivo, whereas overexpressed SHOX2 had the opposite effects. Conclusion: The combination of SHOX2 and RASSF1A DNA methylation had a diagnostic value in pulmonary nodules and early LC. SHOX2 positively modulated the tumorigenesis and metastasis of LC by regulating DNA methylation processes. [ABSTRACT FROM AUTHOR]

Purpose: Diet-related factors are of great significance in the regulation of hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonad (HPG) axes. In this study, we aimed to investigate the effects of chronic exposure to a high fat diet (HFD), fructose or sucralose on the endocrine functions. Methods: Male, Sprague-Dawley rats received a normal chow diet, HFD, 10% fructose or 0.02% sucralose for 10 weeks. Behavioral changes were assessed by open field (OFT) and elevated plus-maze (EPM) tests at week 8. H&E staining was used to observe pathological changes in adrenal cortex, testis and perirenal adipose tissue. Serum hormone concentrations were quantified via enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of genes along the HPA and HPG axes were determined using real-time PCR. Results: All types of dietary interventions increased body weight and disturbed metabolic homeostasis, with anxiogenic phenotype in behavioral tests and damage to cell morphology of adrenal cortex and testis being observed. Along the HPA axis, significantly increased corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) concentrations were observed in the HFD or 0.02% sucralose group. For HPG axis, gonadotropin-releasing hormone (GnRH) and estradiol (E2) concentrations were significantly increased in all dietary intervention groups, while decreased concentrations of follicle-stimulating hormone (FSH) and testosterone (T) were also detected. Moreover, transcriptional profiles of genes involved in the synthesis of hormones and corresponding hormone receptors were significantly altered. Conclusion: Long-term consumption of HFD, fructose or sucralose manifested deleterious effects on endocrine system and resulted in the dysregulation of HPA and HPG axes. [ABSTRACT FROM AUTHOR]

Introduction: Although microRNA (miR)-150-5p participates in the progression of renal fibrosis, its mechanism of action remains elusive. Methods: A mouse model of unilateral ureteral obstruction was used. The in vitro renal fibrosis model was established by stimulating human kidney 2 (HK-2) cells with transforming growth factor beta 1 (TGF-β1). The expression profiles of miR-150-5p, zinc finger E-box binding homeobox 1 (ZEB1), and other fibrosis- and epithelial-mesenchymal transition (EMT)-linked proteins were determined using Western blot and quantitative reverse transcription polymerase chain reaction. The relationship between miR-150-5p and ZEB1 in HK-2 cells was confirmed by a dual-luciferase reporter assay. Results: Both in vivo and in vitro renal fibrosis models revealed reduced miR-150-5p expression and elevated ZEB1 level. A significant decrease in E-cadherin levels, as well as increases in alpha smooth muscle actin (α-SMA) and collagen type I (Col-I) levels, was seen in TGF-β1-treated HK-2 cells. The overexpression of miR-150-5p ameliorated TGF-β1-mediated fibrosis and EMT. Notably, miR-150-5p acts by directly targeting ZEB1. A significant reversal of the inhibitory impact of miR-150-5p on TGF-β1-mediated fibrosis and EMT in HK-2 cells was observed upon ZEB1 overexpression. Conclusion: MiR-150-5p suppresses TGF-β1-induced fibrosis and EMT by targeting ZEB1 in HK-2 cells, providing helpful insights into the therapeutic intervention of renal fibrosis. [ABSTRACT FROM AUTHOR]

Watermelon is a highly cultivated fruit crop renowned for its quality properties of fruit flesh. Among various quality factors, fruit flesh firmness is a crucial quality parameter influencing the fruit texture, shelf life and its commercial value. The auxin/indole-3-acetic acid (Aux/IAA) plays a significant role in fruit development and ripening of non-climacteric fruits. However, the regulatory mechanism of Aux/IAA in controlling fruit flesh firmness and ripening in watermelon remains unknown. In this study, we employed an integrative approach combining genome-wide association study (GWAS) and bulked segregant RNA-Seq analysis (BSR-Seq) to identify an overlapping candidate region between 12 776 310 and 12 968 331 bp on chromosome 6, underlying an auxin-responsive gene (Aux/IAA) associated with flesh firmness in watermelon. Transcriptome analysis, followed by real-time quantitative reverse transcription PCR (qRT-PCR), confirmed that the expression of Aux/IAA was consistently higher in fruits with high flesh firmness. The sequence alignment revealed a single base mutation in the coding region of Aux/IAA. Furthermore, the concomitant Kompetitive/Competitive allele-specific PCR (KASP) genotyping data sets for F2 population and germplasm accessions identified Aux/IAA as a strong candidate gene associated with flesh firmness. Aux/IAA was enriched in the plant hormone signal transduction pathway, involving cell enlargement and leading to low flesh firmness. We determined the higher accumulation of abscisic acid (ABA) in fruits with low flesh firmness than hard flesh. Moreover, overexpression of Aux/IAA induced higher flesh firmness with an increased number of fruit flesh cells while reducing ABA content and flesh cell sizes. Additionally, the allelic variation in Aux/IAA for soft flesh firmness was found to exist in Citrullus mucosospermus and gradually fixed into Citrullus lanatus during domestication, indicating that soft flesh firmness was a domesticated trait. These findings significantly enhanced our understanding of watermelon fruit flesh firmness and consequently the watermelon fruit quality. [ABSTRACT FROM AUTHOR]

This research aims to examine the impact of programmed cell death 4 (PDCD4), microRNA‐21 (miR‐21) and microRNA‐208a (miR‐208a) transcripts, and protein levels on oral epithelial dysplasia (OED) development in oral squamous cell carcinoma (OSCC). Methods: The research investigation involved the collection of saliva, blood, and tissue samples from a total of 20 patients diagnosed with OSCC, 15 patients diagnosed with OED, and 15 healthy individuals. PDCD4, miR‐21, and miR‐208a expression was performed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). PDCD4 protein levels were assessed using enzyme‐linked immunosorbent assay (ELISA) in both saliva and blood samples. For statistical analysis, the Kruskal–Wallis test and the Spearmen rank test were utilised. Results: PDCD4 expression levels were considerably lower in patients with OSCC and OED (p < 0.05) in three biological samples. In contrast, miR‐21 expression was higher in OED and OSCC patients. Patients with low PDCD4 mRNA levels and strong miR‐21 expression had a significant connection (p < 0.05) with tumor size and depth. Conclusions: Examining PDCD4 and miR‐21 transcript levels may help detect the transition from OED to OSCC. This work suggests that PDCD4 and miR‐21 expression levels in liquid biopsies may be biomarkers for OED monitoring in the future. [ABSTRACT FROM AUTHOR]

Introduction: Parkinson's Disease (PD) is a neurodegenerative disorder distinguished from other neurodegenerative disorders by the loss of dopaminergic neurons in the substantia nigra region of the brain, and is the most common neurodegenerative disorder, along with Alzheimer's Disease. PD is characterized by the presence of Lewy bodies when evaluated pathologically. Recent studies showed that the incidence of PH development as a result of genetic mutations alone is very low among all PD cases, and that environmental effects contribute significantly to the disease progression. The molecular mechanisms of diseases are associated with the maintenance of gene and protein expressions as a result of epigenetic regulations. The role of these regulations in the development and pathogenesis of neurodegenerative diseases is still not clearly understood. Methods: In our study, we examined the expression levels of H3C1, H3C12, HDAC4, HDAC5, ANKRD11, ANKRD12, ITM2B and GABBR1, which are genes involved in epigenetic processes in patients with idiopathic PD. Seventy five patients diagnosed with idiopathic PD and 50 healthy controls were included in the study. Peripheral Blood Mononuclear Cell (PBMC) was obtained from whole blood taken from the patient and control groups, and then total RNA was isolated from PBMC. Results: According to the comparison of the patient and control groups, the expression of H3C1, H3C12, ITM2B was high, and the expression of ANKRD11, HDAC4, HDAC5 and GABBR1 was low (p<0.05). Conclusion: As conclusion, we propose that histone regulation is one of the epigenetic mechanisms related to the presence of PD. [ABSTRACT FROM AUTHOR]

Introduction: In this study, we aimed to investigate the clinical effects of COVID-19 infection and vaccines on Myasthenia gravis (MG) during the pandemic. Methods: A total of 141 MG patients between April 2020 and December 2021 were retrospectively analyzed. Data including demographic and clinical characteristics of patients, COVID-19 test results, and vaccine types (mRNA-BNT162b2 and/or inactivated-CoronaVac) were recorded. All patients were followed by face-to-face interviews and/or phone calls. Worsening MG symptoms after COVID-19 infection or vaccines were noted. Results: A total of 60 patients were diagnosed with COVID-19, and reverse transcriptase-polymerase chain reaction test results were COVID-19 positive in 54 (90%) patients. Twenty-eight (46.7%) patients had lung involvement, while 20(33.3%) patients were followed in the ward. Twelve (20%) patients were followed in the intensive care unit, and two of them (3.3%) died. Both deceased patients were unvaccinated. The most common symptoms were fatigue (78.3%), and 13(21.7%) patients were asymptomatic. Of the patients, 96(68%) received at least one dose BNT162b2 or CoronaVac, while 30.4% of the patients received ≥3 doses of vaccines. The local skin irritation and fatigue rate was significantly higher with BNT162b2 vaccine than CoronaVac (p<0.001 and p=0.004, respectively). No serious side effect was observed with either vaccine. Five patients had worsening MG symptoms after vaccination during a six-week follow-up. None of the patients experienced myasthenic crises. Conclusion: Our study results suggest that COVID-19 infection affects MG similar to the general population and does not lead to worsening MG symptoms. Both mRNA and inactivated vaccines with proven efficacy can be used safely in MG patients. [ABSTRACT FROM AUTHOR]

A nationwide cohort study investigated the association between prior respiratory syncytial viral (RSV) infection and the severity of COVID-19. The study found that individuals with a history of RSV infection had a higher susceptibility to COVID-19, increased risk of hospitalization, and greater severity of the disease. This association was particularly pronounced in older individuals and those with underlying conditions such as diabetes and congestive heart failure. The study suggests that recent RSV infection contributes to the burden of COVID-19 and highlights the potential benefits of RSV vaccination in mitigating the clinical course of COVID-19. [Extracted from the article]

Human enteric viruses, such as hepatitis A virus (HAV), hepatitis E virus (HEV), and norovirus genogroups I and II (NoVGI and NoVGII), cause infections, and it has been largely demonstrated that mussels play an important role if consumed as raw or undercooked food matrices. This study aimed to investigate, through qualitative and quantitative biomolecular assays, the detection of partial genomic regions belonging to the most relevant enteropathogenic viruses for humans (HAV, HEV, NoVGI and NoVGII) in mussels (Mytilus galloprovincialis) farmed along the coasts of two Italian regions on the central Adriatic Sea: Abruzzo (Casalbordino, Chieti) and Molise (Termoli, Campobasso). A total of 425 animals were sampled, and the respective georeferentiations were registered. A total of 85 pools, each composed of five subjects/aliquots, were formed (22 from Abruzzo and 63 from Molise regions). This step was followed by homogenization and RNA extraction, and then the biomolecular assays [nested reverse transcription polymerase chain reaction (PCR) and real-time reverse transcription-quantitative PCR] were performed. 1.17% of the pool was positive for HAV RNA detection (10² copies/mL), 9.41% for HEV (10²-10³ copies/µL), 2.35% for NoVGI (10¹ copies/µL), and no pool was positive for NoVGII. This study demonstrated the human enteric viruses' presence in mussels farmed in a low-investigated marine area. Based on a one-health point of view, this paper aims to enforce the importance of biomolecular and epidemiological screenings as surveillance systems to guarantee human, animal, and environmental health. [ABSTRACT FROM AUTHOR]

Background: The relationship between erectile dysfunction (ED) and type 1 diabetes mellitus (T1DM) is currently a hot topic of medical research. It has been reported that autophagy plays a crucial role in causing erectile dysfunction in T1DM. Recent research has shown that mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2) is strongly linked to the development of T1DM. However, the specific mechanism by which it regulates the erectile function is not yet fully understood. Objectives: To investigate whether HMGCS2 affects erectile function in type 1 diabetic rats by regulating autophagy in corpus cavernosum endothelial cells (CCECs). Materials and methods: First, the rat model of T1DM was established. Then, the ratio of maximum penile intracavernous pressure (ICPmax) and mean arterial pressure (MAP) was detected to assess the erectile function in various groups, and the protein expression of HMGCS2, mTOR and p‐mTOR was evaluated by western blot (WB) and immunohistochemistry (IHC). To explore the relationship between HMGCS2 and the mTOR signaling pathway in T1DM ED rats, we silenced the expression of HMGCS2 and activated the mTOR signaling pathway with MHY1485 in CCECs and then assessed the expression of beclin1, P62, LC3, autophagosome, endothelial nitric oxide synthase (eNOS), phosphorylation of eNOS (p‐eNOS), and nitric oxide (NO) to evaluate autophagy and the erectile function by reverse transcription quantitative polymerase chain reaction and western blot. Results: The study conducted on T1DM ED rats showed that the expression of HMGCS2 was significantly increased, while the autophagy was suppressed. Additionally, the mTOR signaling pathway was highly activated. In contrast, when HMGCS2 was silenced in vitro, p‐mTOR/mTOR was reduced, and autophagy was improved. These effects were accompanied by the enhanced activity of eNOS. Furthermore, when HMGCS2 was silenced and the mTOR signaling pathway was simultaneously activated, the results revealed a decrease in autophagy as well as a reduction in activity of eNOS in comparison to just silencing HMGCS2 alone. Discussion and conclusion: HMGCS2 upregulation in T1DM rats inhibited autophagy and eNOS activity by activating the mTOR pathway and led to a decrease in the erectile function. [ABSTRACT FROM AUTHOR]

Eimeria bovis is a pathogenic protozoan that causes cattle digestive tract infections, which can cause economic losses to farmers. It is necessary to develop specific and accurate detection methods to conserve livestock and prevent coccidiosis in Indonesia. This study aims to detect E. bovis by nested PCR and determine the relationship with reference sequences. A total of 167 samples of beef cattle feces were taken randomly from community farms spread across 18 provinces in Indonesia. The feces were examined natively, and then the oocysts were purified by the sugar flotation method, extracted by KIT extraction, and amplified by the nPCR technique. Positive samples were followed by sequencing and phylogenetic analysis using MEGA 11 software. This study used two pairs of primers (outer and inner) taken from ITS-1 molecular markers. As many as 96 out of 167 samples (57.5%) were positive for Eimeria spp., and 48 of the 96 samples were positive for Eimeria spp. (50%) were detected to be positive for E. bovis based on the presence of a 238 bp DNA fragment. The results of the phylogenetic analysis showed that the study sample formed a separate cluster from the E. bovis cluster from abroad. In conclusion, E. bovis was detected in 16 out of 18 provinces in this study, and the nPCR technique proved to have better sensitivity and specificity. [ABSTRACT FROM AUTHOR]

The association of air pollution and greenspace with respiratory pathogen acquisition and respiratory health was investigated in a community-based birth-cohort of 158 Australian children. Weekly nasal swabs and daily symptom-diaries were collected for 2-years, with annual reviews from ages 3-7-years. Annual exposure to fine-particulate-matter (PM2.5), nitrogen-dioxide (NO2), and normalised-difference-vegetation-index (NDVI) was estimated for pregnancy and the first 2-years-of-life. We examined rhinovirus, any respiratory virus, Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae detections in the first 3-months-of-life, age at initial pathogen detection, wheezing in the first 2-years, and asthma at ages 5-7-years. Our findings suggest that higher NDVI was associated with fewer viral and M. catarrhalis detections in the first 3-months, while increased PM2.5 and NO2 were linked to earlier symptomatic rhinovirus and H. influenzae detections, respectively. However, no associations were observed with wheezing or asthma. Early-life exposure to air pollution and greenspace may influence early-life respiratory pathogen acquisition and illness. [ABSTRACT FROM AUTHOR]

Background: Type-2 diabetes mellitus is a metabolic disorder characterised by hyperglycemia and insulin resistance. This study aims to explore the role and mechanism of DIRAS family GTPase 3 (DIRAS3) in mediating pancreatic β-cell death and insulin secretion. Materials and Methods: A bioinformatic analysis of the GSE118230 and GSE150281 datasets was performed to screen differentially expressed genes. The pancreatic β-cell lines, INS-1 and MIN6, were treated with palmitic acid (PA) to mimic the cell models of type-2 diabetes mellitus. CCK-8 assay, 5-ethynyl-2'-deoxyuridine staining, flow cytometry, enzyme-linked immunoassay, immunofluorescence, qRT-PCR, and western immunoblotting were conducted to illustrate the role of DIRAS3 in the cell models. Results: Unlike in normal controls, DIRAS3 was highly expressed in PA-treated pancreatic β-cells in a dose- and time-dependent manner. Moreover, the silencing of DIRAS3 in the INS-1 cells attenuated PA-induced cell loss by improving cell proliferation and inhibiting apoptosis and prevented the PA-induced impairment of insulin secretion. Consistently, the overexpression of DIRAS3 in the MIN6 cells accelerated PA-induced cell loss and impaired insulin secretion. A Gene Set Enrichment Analysis predicted that phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) was a downstream signalling pathway of DIRAS3, as the inhibitory effects of DIRAS3 on the activation of the PI3K/AKT signalling pathway were confirmed in the INS-1 and MIN6 cells. Moreover, the PI3K inhibitor, LY294002, effectively reversed the protective effects of DIRAS3 silencing on the INS-1 cells. Conclusion: DIRAS3 was highly expressed in the cell models of type-2 diabetes mellitus, contributing to PA-induced cell death and impaired insulin secretion in pancreatic β-cells through the inhibition of the PI3K/AKT signalling pathway. [ABSTRACT FROM AUTHOR]

Netrin-4 (NTN4), a secreted protein from the Netrin family, has been recognized for its role in vascular development, endothelial homeostasis and angiogenesis. Vascular endothelial (VE)-cadherin is a specialized adhesion protein located at the intercellular junctions of endothelial cells (ECs), and regulates migration, proliferation and permeability. To date, the relationship between NTN4 and VE-cadherin in ECs remains unclear. In the present study, human umbilical vein ECs (HUVECs) were transfected with NTN4 overexpression plasmid, resulting in NTN4 overexpression. Reverse transcription-quantitative PCR and western blotting were used to determine gene and protein expression. CCK8, wound healing, and Transwell assays were performed to evaluate cell proliferation, migration and permeability. NTN4 overexpression decreased HUVEC viability and migration. In addition, NTN4 overexpression increased the expression of VE-cadherin and decreased the permeability of HUVECs. Subsequent studies showed that NTN4 overexpression increased the NF-κB protein level and decreased IκB-α protein expression in HUVECs. In HUVECs treated with NF-κB inhibitor pyrrolidine dithiocarbamate, the expression of VE-cadherin failed to increase with NTN4 overexpression. Taken together, the results indicated that NTN4 overexpression increased VE-cadherin expression through the activation of the NF-κB signaling pathway in HUVECs. The present findings revealed a novel regulatory mechanism for VE-cadherin expression and suggested a novel avenue for future research on the role of NTN4 in endothelial barrier-related diseases. [ABSTRACT FROM AUTHOR]

Ischemic heart disease (IHD) remains a leading cause of mortalities worldwide, necessitating timely reperfusion to reduce acute mortality. Paradoxically, reperfusion can induce myocardial ischemia/reperfusion (I/R) injury, which is primarily characterized by mitochondrial dysfunction. Translocator protein (TSPO) participates in multiple cellular events; however, its role in IHD, especially in the process of myocardial I/R injury, has not been well determined. The aim of the present study was to investigate the functional role of TSPO in myocardial I/R injury and dissect the concomitant cellular events involved. This study utilized small interfering RNA (siRNA) technology to knock down TSPO expression. The I/R process was simulated using an anoxia/reoxygenation (A/R) model. The role of TSPO in H9c2 cardiomyocytes was assessed using various techniques, such as Western blotting, Flow cytometry, Reverse transcription-quantitative PCR (RT-qPCR), Immunofluorescence, Co-immunoprecipitation (co-IP) and similar methods. It was found that A/R markedly upregulated the expression of TSPO in cardiomyocytes. Inhibition of TSPO improved myocardial cell apoptosis and damage following A/R stimulation. Additionally, targeting TSPO alleviated mitochondrial damage, reduced mitochondrial ROS release and enhanced ATP synthesis following A/R stimulation. It was further confirmed that A/R stimulation induced a significant increase in the expression of pivotal markers [phosporylated-PKR-like ER kinase (PERK)/PERK, activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1] involved in the adaptive unfolded protein response, which is accompanied by downstream signaling during endoplasmic reticulum (ER) stress. Notably, TSPO knockdown increased the expression of the aforementioned markers and, subsequently, TSPO was confirmed to interact with ATF6, suggesting that TSPO might play a role in ER stress during myocardial I/R injury. Finally, inhibition of TSPO upregulated mitophagy, as indicated by further decreases in P62 and increases in Parkin and PINK1 levels following A/R stimulation. Together, the results suggest that TSPO plays a multifaceted role in myocardial I/R injury. Understanding TSPO-induced cellular responses could inform targeted therapeutic strategies for patients with IHD. [ABSTRACT FROM AUTHOR]

Background Psoriasis is a T cell-mediated chronic inflammatory skin condition characterized by the interaction of T cells with various cell types, forming an inflammatory microenvironment that sustains psoriatic inflammation. Homeostasis of these tissue-resident T cells is supported by fibroblasts, the primary structural cells in the dermis. In psoriasis, there is increased expression of matrix metalloproteinase 2 (MMP2), mediating structural alterations in skin tissues and modulating inflammation. Additionally, the CD100–plexin-B2 (PLXNB2) axis is known to enhance psoriasis inflammation via keratinocytes, and CD103 levels are associated with the severity of psoriasis upon relapse. Objectives To elucidate the role of fibroblasts and the MMP2–CD100 axis in modulating psoriasis inflammation. Methods CD100 expression and function in psoriasis were assessed using immunofluorescence, enzyme-linked immunosorbent assay, single-cell transcriptome sequencing, cellular interaction analyses and quantitative reverse transcriptase polymerase chain reaction. CD8+ T cells from people with psoriasis were isolated using magnetic beads, to investigate the regulatory effect of MMP2 on CD100 expression on their membranes. Single-cell transcriptome sequencing, spatial transcriptome sequencing, mimetic timing analysis, immunofluorescence and flow cytometry were used to determine the origin of MMP2 and its impact on CD103+ CD8+ T cells. The hypotheses were further validated in vivo using MMP2 and CD100 inhibitors. Results Soluble CD100 (sCD100) was significantly upregulated in both psoriatic lesions and peripheral blood, amplifying psoriasis inflammation by promoting the production of inflammatory cytokines by keratinocytes, fibroblasts and endothelial cells via the sCD100–PLXNB2 axis. Fibroblasts that highly expressed MMP2 (MMP2hi) exacerbated psoriasis symptoms by facilitating CD100 shedding from CD8+ T-cell membranes. Additionally, it was shown that fibroblasts enhance the upregulation of the CD8+ T-cell residency factor CD103 in co-cultures with CD8+ T cells. Inhibitors targeting MMP2 and CD100 were effective in reducing inflammation in an imiquimod-induced psoriasis model. Conclusions Our findings underscore the pivotal role of MMP2hi fibroblasts in the amplification and recurrence of inflammatory responses in psoriasis. These fibroblasts augment psoriasis inflammation through the CD100–PLXNB2 axis by facilitating CD100 shedding on CD8+ T-cell membranes and by upregulating CD103, thereby enhancing CD8+ T-cell residency. [ABSTRACT FROM AUTHOR]

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter required for excitation/inhibition balance is synthesized by the glutamic acid decarboxylases (GADs) in GABAergic neurons. The levels and activity of GADs are strongly correlated with GABA and neural transmission. Dysregulation of GADs and GABA is associated with various neurological disorders. The study used psoralidin, found in the seeds of Psoralea corylifolia, to investigate its effect on GAD levels and regulatory mechanisms in primary cortical neurons. Psoralidin reduced GAD67 through transcriptional regulation. The reduction was not mediated by the N-methyl-D-aspartate receptor. Additionally, psoralidin attenuated the formation of inhibitory synapses in primary hippocampal neurons. [ABSTRACT FROM AUTHOR]

Globally, there is a move towards using local, native species for ecotoxicological risk assessments. Anthropogenic stressors from urban, agricultural, and industrial activities can impact the health of receiving ecosystems. Biomarkers can provide valuable insights as early warning signals of the potential environmental impacts of stressors. The aim of this study was to develop biomarkers in the green-lipped mussel (Perna canaliculus), a potential bioindicator of environmental health for coastal marine ecosystems in New Zealand. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays targeting the expression of genes involved in oxidative stress, xenobiotic transfer, membrane transportation, cellular and DNA response/repair, and endocrine disruption were developed and validated for P. canaliculus. We found significant modulation of genes associated with oxidative stress, xenobiotic transfer, membrane transport, endocrine disruption, and genotoxicity in P. canaliculus following 48-hour exposures to copper and benzo[α]pyrene. These results demonstrate the potential of P. canaliculus as a bioindicator species for environmental risk assessment. The gene expression assays showed potential as early indicators of exposure to the chemicals tested but require additional validation to assess their ability to predict effects at higher levels of biological organisation. [ABSTRACT FROM AUTHOR]

Copyright of Arquivos Brasileiros de Oftalmologia is the property of Arquivos Brasileiros de Oftalmologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background & Objective: The study aims to determine the adverse effects of a mild COVID-19 infection on maternal and neonatal outcomes in pregnant women living in the eastern part of Tehran. Materials & Methods: In this prospective cohort study, we followed two groups of pregnant women until term and their pregnancy and neonatal outcomes. RT-PCR (reverse transcriptase polymerase chain reaction) positive tests of nasopharyngeal swabs were confirmed in one group, where at least three signs and symptoms associated with COVID-19, and/or chest CT scans identified highly specific findings for the disease were present. The control group is composed of healthy pregnant women tested negative for COVID-19. Results: The study included 319 pregnant women (108 with mild COVID-19 infection and 211 healthy). A higher percentage of mothers with mild COVID-19 infection had preterm births (47.9% vs. 30%, p=0.007), and their neonates required hospitalization more often (45.8% vs. 22.6%, p<0.001). PTB was positively associated with maternal hypertension (relative risk (RR): 5.40), mild COVID-19 infection (RR: 2.05), and older age (RR: 1.05). Among the risk factors for neonatal hospitalization, PTB (RR: 5.94), maternal hypertension (RR: 2.74), and mild COVID-19 infection during pregnancy (RR: 2.57) were significant. Conclusion: The outcomes of pregnancy are significantly impacted by maternal infection with COVID-19, even if it is mild. Preterm births are more likely to happen in mothers with mild COVID-19 infection, and neonates need more hospitalizations. [ABSTRACT FROM AUTHOR]

Copyright of Spor Hekimligi Dergisi/Turkish Journal of Sports Medicine is the property of Turkish Journal of Sports Medicine / Spor Hekimleri Dernegi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background The 2022 African Cup of Nations (AFCON) took place in Cameroon from January 9th to February 5th, 2022, including Garoua in the north. We aimed to measure the impact of this event on the local COVID-19 epidemic given the implementation of a preventive strategy based on a health pass. Methods All players, staff and fans involved in the AFCON event were screened with PCR tests. Symptomatic cases were also continuously monitored in the general population and screened for variants of concern. Daily numbers of confirmed cases were compared to neighboring countries numbers retrieved from a public domain source. Results In total, 1479 and 2481 tests were performed in the general population and on asymptomatic AFCON attendees, respectively. From the latter, 12.5% were PCR-positive; 97% were infected with Omicron, with no significant difference compared to the passive program (G-test, P value = 0.162). Surveillance indicated the AFCON did not increase the number of symptomatic PCR-positive cases in the general population compared to neighboring countries. Conclusions Though the COVID-19 epidemic was fueled by asymptomatic cases infected with the Omicron variant at the time, the non-therapeutic preventive measures implemented for AFCON mitigated an increase in the epidemic in the local population. [ABSTRACT FROM AUTHOR]

Gliomas are the most prevalent primary malignant brain tumors worldwide. Growing evidences indicate that circular RNAs (circRNAs) play an important role in the regulation of biological behavior of tumors. We aimed to investigate the role and mechanism of circVCAN in glioma. RNase R treatment was utilized to assess the cyclic properties of circVCAN. CircVCAN, miR‐488‐3p, and myocyte enhancer factor 2C (MEF2C) levels in glioma tissues and cells were detected by reverse transcription real‐time polymerase chain reaction (RT‐qPCR), and the localization of them in glioma cells was determined with fluorescence in situ hybridization. Furthermore, a variety of biologically functional assessments were used to validate the role of circVCAN in glioma. The regulatory mechanisms of circVCAN, miR‐488‐3p, and MEF2C were further confirmed by double luciferase reporter gene assay, RNA immunoprecipitation and RNA pull‐down assay, and the binding of MEF2C to JAGGED1 was revealed by chromatin immunoprecipitation. Additionally, a xenograft tumor model was constructed to demonstrate the effect of circVCAN on tumor growth in vivo. Our results indicated that circVCAN was more stable than its linear RNA and was significantly upregulated in gliomas. CircVCAN overexpression stimulated glioma cells to proliferate and metastasize, but circVCAN silencing exerted the opposite effect. Meanwhile, silencing circVCAN inhibited tumor growth in vivo. Moreover, we found that circVCAN interacted with miR‐488‐3p to regulate MEF2C expression, and miR‐488‐3p inhibition or MEF2C overexpression reversed the inhibitory effect on malignant bio‐behaviors mediated by circVCAN knockdown in glioma cells. MEF2C promoted the transcription of JAGGED1, and circVCAN knockdown reduced the binding between MEF2C and JAGGED1. Collectively, circVCAN is a carcinogenic circRNA in glioma, and the circVCAN/miR‐488‐3p/MEF2C‐JAGGED1 axis could serve as a potential target for the management of glioma. [ABSTRACT FROM AUTHOR]

Background & aims: Perilipin 1 (PLIN1) is an essential lipid droplet surface protein that participates in cell life activities by regulating energy balance and lipid metabolism. PLIN1 has been shown to be closely related to the development of numerous tumor types. The purpose of this work was to elucidate the clinicopathologic significance of PLIN1 in hepatocellular carcinoma (HCC), as well as its impact on the biological functions of HCC cells, and to investigate the underlying mechanisms involved. Methods: Public high-throughput RNA microarray and RNA sequencing data were collected to examine PLIN1 levels and clinical significance in patients with HCC. Immunohistochemistry (IHC) and real-time quantitative reverse transcription polymerase chain reaction (RT‒qPCR) were conducted to assess the expression levels and the clinicopathological relevance of PLIN1 in HCC. Then, SK and Huh7 cells were transfected with a lentivirus overexpressing PLIN1. CCK8 assay, wound healing assay, transwell assay, and flow cytometric analysis were conducted to explore the effects of PLIN1 overexpression on HCC cell proliferation, migration, invasion, and cell cycle distribution. Ultimately, Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate the underlying mechanisms of PLIN1 in HCC progression based on HCC differentially expressed genes and PLIN1 co-expressed genes. Results: PLIN1 was markedly downregulated in HCC tissues, which correlated with a noticeably worse prognosis for HCC patients. Additionally, PLIN1 overexpression inhibited the proliferation, migration, and invasion of SK and Huh7 cells in vitro, as well as arresting the HCC cell cycle at the G0/G1 phase. More significantly, energy conversion-related biological processes, lipid metabolism, and cell cycle signalling pathways were the three most enriched molecular mechanisms. Conclusion: The present study revealed that PLIN1 downregulation is associated with poor prognosis in HCC patients and accelerated HCC progression by promoting cellular proliferation, migration, and metastasis, as well as the mechanisms underlying the regulation of lipid metabolism-related pathways in HCC. [ABSTRACT FROM AUTHOR]

Antimicrobial photodynamic therapy (aPDT) has shown efficacy in inactivating different bacterial species by photosensitizer-induced free radical production. Despite aPDT is considered unable to cause resistant strains, enzymatic pathways for detoxification of reactive oxygen species and transmembrane photosensitizer efflux systems could cause resistance to aPDT. Resistance mechanisms can be evaluated by measurement of mRNA from by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Thus, the aim of this study was to access the mRNA level data obtained by RT-qPCR in bacterial cells submitted to photodynamic therapy. Studies performed on mRNA levels in bacteria after PDT were assessed on MEDLINE/Pubmed. The mRNA levels from genes related to various functions have been successfully evaluated in both Gram-positive and -negative bacteria after aPDT by RT-qPCR. Such an approach has improved the understanding of aPDT-induced effects, and reinforced the effectiveness of aPDT on bacteria, which can cause infections in different human tissues. [ABSTRACT FROM AUTHOR]

Background: The significance of CXCL5 in pancreatic cancer is unclear, although it has been implicated in the malignant process of many different types of cancer. Research on the impact of CXCL5 on immune cell infiltration and the malignant phenotype of pancreatic cancer is needed. This study aimed to examine the connection between CXCL5 expression and immune cell infiltration and the malignant phenotype of pancreatic cancer. Methods: Tissue samples and clinical information were collected from 90 patients with pancreatic cancer. Tumour tissues and adjacent tissues were made into a tissue microarray and stained for immunohistochemistry analysis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to measure the expression level of CXCL5. CXCL5-overexpressing/CXCL5-knockdown cell lines were constructed via transfection for cytological experiments. CCK-8, cell apoptosis, cell cycle, cell invasion, and cell colony formation assays were used to detect the effect of CXCL5 on the malignant phenotype of pancreatic cancer cells. Finally, a mouse model of pancreatic cancer was constructed for in vivo verification. Results: Compared with control cells, pancreatic cancer cells overexpressing CXCL5 exhibited increased proliferation, migration, and invasion but decreased apoptosis. Conversely, knockdown of CXCL5 did not enhance the malignant phenotype of pancreatic cancer cells. Spearman correlation analysis indicated that there was a significant negative correlation between CXCL5 levels and the CD8 IRS. However, there was a significant positive correlation between FOXP3 IRS and CXCL5 levels. Conclusions: CXCL5 is highly expressed in pancreatic cancer and promotes the malignant phenotype of pancreatic cancer cells. CXCL5 is associated with immunosuppressive FOXP3 + T-cell infiltration, which facilitates the formation of an immunosuppressive microenvironment (with low CD8 + T-cell infiltration). [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) becomes a fatal gynecologic malignant cancer in females worldwide. Target therapy is a promising therapeutical choice for patients with OC, and identifying biomarkers and exploring molecular mechanisms are necessary. In this study, the functions and mechanism of long noncoding RNA transient receptor potential cation channel subfamily M member 2 antisense RNA (TRPM2-AS) in OC were explored. TRPM2-AS expression in OC cells was analyzed utilizing reverse transcription quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) and colony forming assays were carried out to explore the influence of TRPM2-AS on OC cell viability and proliferation. Cell apoptosis was detected using TdT-mediated dUTP Nick-End labeling (TUNEL) and flow cytometry analysis. Protein expression of apoptotic markers was subjected to western blotting. RNA pulldown or luciferase reporter assays were applied to explore the interaction between TRPM2-AS and miR-6764-5p or the binding of miR-6764-5p and TRPM2. The results showed that TRPM2-AS is highly expressed in OC cells and was mainly localized in cytoplasm. TRPM2-AS depletion suppressed OC cell viability and proliferation while increasing cell apoptotic rate. TRPM2 displayed a high level in OC cells and was positively regulated by TRPM2-AS. TRPM2-AS interacted with miR-6764-5p and thereby upregulated TRPM2 expression. In addition, TRPM2 overexpression reversed the repressive impact of TRPM2-AS depletion on malignant OC cellular process. In conclusion, TRPM2-AS promotes OC cell viability and proliferation while enhancing cell apoptosis through interaction with miR-6764-5p to regulate TRPM2 level. [ABSTRACT FROM AUTHOR]

Background: Open fractures are challenging due to susceptibility to Staphylococcus aureus infections. This study examines the impact of Vancomycin-Loaded Calcium Sulfate (VLCS) and negative pressure wound therapy (NPWT) on macrophage behavior in enhancing healing and infection resistance. Both VLCS and NPWT were evaluated individually and in combination to determine their effects on macrophage polarization and infection resistance in open fractures. Methods: Through single-cell RNA sequencing, genomic expressions in macrophages from open fracture patients treated with VLCS and NPWT were compared to a control group. The analysis focused on MBD2 gene changes related to macrophage polarization. Results: Remarkable modifications in MBD2 expression in the treatment group indicate a shift towards M2 macrophage polarization. Additionally, the combined treatment group exhibited greater improvements in infection resistance and healing compared to the individual treatments. This shift suggests a healing-promoting atmosphere with improved infection resilience. Conclusions: VLCS and NPWT demonstrate the ability to alter macrophage behavior toward M2 polarization, which is crucial for infection prevention in open fractures. The synergistic effect of their combined use shows even greater promise in enhancing outcomes in orthopedic trauma care. [ABSTRACT FROM AUTHOR]

Background: Tocotrienol is a vitamin E analogue that is known to exert anti-inflammatory and antioxidant effects. Hence, in the current study, the effects of TRF on the expression of pro- and anti-apoptotic proteins in the streptozotocin-induced diabetic rat retinas were investigated. The effect of TRF on the visual behaviour of rats was also studied. Methods: Diabetes was induced in rats by intraperitoneal injection of streptozotocin and was confirmed by a blood sugar level of at least 20 mmol/L, 48 h, post-injection. Diabetic rats were divided into a group treated with vehicle (DV) and the other treated with TRF (100 mg/kg; DT). A group of non-diabetic rats treated with vehicle (N) served as the control group. All treatments were administered orally for 12 weeks. Rats were then subjected to an assessment of general behaviour in an open field arena and a two-chamber mirror test to assess their visual behaviour. At the end of the experimental period, rats were sacrificed, and their retinas were isolated to measure the expression of pro- (Casp3, Bax) and anti-apoptotic (Bcl2) markers using RT-qPCR and ELISA. TUNEL staining was used to detect the apoptotic retinal cells. Results: Treatment with TRF lowered the retinal expression of Casp3 protein by 2.26-folds (p < 0.001) and Bax protein by 2.18-fold (p < 0.001) compared to vehicle-treated rats. The retinal anti-apoptotic protein Bcl2 expression was 1.87-fold higher in DT compared to DV rats (p < 0.001). Accordingly, the Bax/Bcl2 ratio in the TRF-treated group was significantly greater in DT compared to DV rats. Retinal Casp3, Bax, and Bcl2 gene expression, as determined by RT-qPCR, also showed changes corresponding to protein expression. In the open field test, DV rats showed greater anxiety-related behaviour than group N, while the behaviour of DT rats was similar to the N group of rats. DT rats and group N rats preferred the inverse mirror chamber over the mirror-containing chamber in the two-mirror chamber test (p < 0.01). Conclusion: Oral TRF therapy for 12 weeks lowers retinal cell apoptosis by decreasing pro- and increasing anti-apoptotic markers. The preservation of visual behaviour in a two-chamber mirror test supported these retinal molecular alterations in diabetic rats. [ABSTRACT FROM AUTHOR]

Background: The number and proportion of HIV/AIDS patients among older people are continuously and rapidly increasing in China. We conducted a detailed molecular epidemiological analysis of HIV-1 epidemic strains in a developed city in eastern China and found that elderly people play a crucial role in the transmission of subtypes and high pretreatment drug resistance (PDR). Methods: A total of 1048 samples were obtained from 1129 (92.8%) newly confirmed HIV-1-positive and treatment-naive patients between 2019 and 2023. The 1316 bp target fragment of the pol gene was amplified by reverse transcription polymerase chain reaction (RT‒PCR) and nested PCR, and Maximum-likelihood (ML) phylogenetic trees and molecular transmission network were constructed to analyse the subtypes and transmission clusters. Molecular transmission network was visualized using Cytoscape with the distance threshold of 0.0075. PDR-associated mutations were determined according to the Stanford University HIV Drug Resistance Database. Results: A total of 933 pol sequences (89.0%, 933/1048) were successfully obtained, and twelve HIV-1 subtypes were detected. CRF07_BC was the predominant subtype, accounting for 48.1% (449/933) of sequences, followed by CRF01_AE (29.4%, 274/933). A total of 398 individuals (42.7%, 398/933) formed 89 clusters in the network. Multivariable logistic regression analysis revealed that age, nationality, subtype, and PDR were the most significant factors associated with clustering in the transmission network. The prevalence of PDR was 14.6% (136/933).PDR associated with non-nucleoside reverse transcriptase inhibitors (10.0%, 93/933) was much more common than that associated with nucleoside reverse transcriptase inhibitors (1.8%, 17/933) and protease inhibitors (3.2%, 30/933) (χ2 = 77.961, p < 0.001). The most frequent NNRTI mutations were K103N/S/KN/NS (52.2%, 71/136), which led to the highest proportion of high-level resistance to nevirapine and efavirenz (52.2%). Conclusions: Our study revealed the important influence of elderly people on CRF07_BC transmission and the high prevalence of PDR. The clustering of drug-resistant cases was significant, which suggested the potential for localized widespread transmission of drug-resistant strains. HIV screening and the determination of PDR are recommended for older patients to improve early detection and reduce treatment failure and second-generation transmission. [ABSTRACT FROM AUTHOR]

Background: Deep vein thrombosis (DVT) of lower extremity is a common complications after total knee arthroplasty (TKA). The purpose of this study was to evaluate the risk factors for DVT after TKA and analyze the expression of miR-199b-5p and nitric oxide (NO) before and after TKA, as well as their predictive value for DVT. Methods: Basic clinical information of 121 patients with TKA was analyzed retrospectively. RT-qPCR was used to detect the relative expression level of miR-199b-5p in patients before and after TKA treatment. Based on the occurrence of DVT, patients were divided into DVT and non-DVT groups. Logistic regression analysis evaluated the risk factors of DVT. The receiver operating characteristic (ROC) curve assessed the predictive value of postoperative miR-199b-5p level, preoperative NO level, and their combination in DVT. The target genes of miR-199b-5p and their functions were predicted and annotated using bioinformatics analysis. Results: The level of miR-199b-5p after TKA was upregulated compared with that before TKA (P < 0.001). DVT occurred in 20 of 121 patients after TKA, with an incidence of 16.53%. Multivariate analysis showed that age, family history of DVT, decrease of NO and increase of miR-199b-5p were risk factors for DVT after TKA (P < 0.05). The ROC curve showed that both miR-199b-5p and NO had certain diagnostic value for DVT, but the combination of miR-199b-5p and NO had the highest diagnostic accuracy (P < 0.001). Conclusion: This study showed that the expression of miR-199b-5p was up-regulated after TKA, and miR-199b-5p levels were higher in DVT patients than in non-DVT patients. miR-199b-5p combined with NO is of great value in the diagnosis of DVT after TKA. [ABSTRACT FROM AUTHOR]

Background: Vascular endothelial growth factor (VEGF) is an important proangiogenic factor and has been considered as a key target of antiangiogenetic therapy in oral squamous cell carcinoma (OSCC). However, clinical application of bevacizumab, a specific VEGF antibody, didn't improve the survival rate of OSCC patients. One possible explanation is that VEGF gene expresses diverse isoforms, which associate with extracellular vesicles (EVs), and EVs potentially contribute to VEGF resistance to bevacizumab. However, clear solution is lacking in addressing this issue. Methods: Expression of VEGF isoforms in OSCC cells was confirmed by reverse transcription and polymerase chain reaction (RT-PCR) and western blot. EVs isolated from OSCC cell's conditioned medium (CM) were characterized by western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunogold labeling and western blot were applied to study the VEGF on EVs. Tube formation assay and Matrigel plug angiogenesis assay were used for analyzing the angiogenesis capacity of EV-VEGF. Results: The most popular isoforms expressed by VEGF gene are VEGF121, VEGF165 and VEGF189. In this study, we demonstrated that all three isoforms of mRNA could be detected at varying levels in OSCC cells, while only VEGF165 and VEGF189 proteins were found. CM derived from OSCC cells, both soluble and non-soluble forms of VEGF could be detected. We further confirmed the presence of VGEF189 bound to EVs as a non-soluble form. EV-bound VEGF189 presented angiogenic activity, which could not be neutralized by bevacizumab. It was found that VEGF189 bound to EVs by heparan sulfate proteoglycans (HSPG). In addition, the angiogenic effect of EV-VEGF could be reversed by surfen, a kind of HSPG antagonist both in vitro and in vivo. Conclusion: Antagonists targeting HSPG might potentially overcome the resistance of EV-VEGF to bevacizumab and serve as an alternative for anti-VEGF therapy in OSCC. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 2 (FGF2) is a crucial factor in odontoblast differentiation and dentin matrix deposition, which facilitates pulpodentin repair and regeneration. Nevertheless, the specific biological function of FGF2 in odontoblastic differentiation remains unclear because it is controlled by complex signalling pathways. This study aimed to investigate the mechanism underlying the effect of FGF2 on osteo/odontogenic differentiation of stem cells from the apical papilla (SCAP). SCAP were pretreated with conditioned media containing FGF2 for 1 week, followed by culturing in induced differentiation medium for another week. RNA sequencing (RNA-seq) combined with quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the pathways affected by FGF2 in SCAP. Osteo/odontogenic differentiation of SCAP was determined using Alizarin red S staining, alkaline phosphatase staining, RT-qPCR, and western blotting. Pretreatment with FGF2 for 1 week increased the osteo/odontogenic differentiation ability of SCAP. RNA-seq and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that phosphatidylinositol 3-kinase (PI3K)/AKT signalling is involved in the osteogenic function of FGF2. RT-qPCR results indicated that SCAP expressed FGF receptors, and western blotting showed that p-AKT was reduced in FGF2-pretreated SCAP. The activation of the PI3K/AKT pathway partially reversed the stimulatory effect of FGF2 on osteo/odontogenic differentiation of SCAP. Our findings suggest that pretreatment with FGF2 enhances the osteo/odontogenic differentiation ability of SCAP by inhibiting the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Background: Regional evidence on the role of vitamin D in the prevention of COVID-19 and its sequalae remains inconclusive and sparse. Objective: This study aimed to determine the association of COVID-19 status and severity with vitamin D among adults in Colombo District, Sri Lanka. Methods: A sex-matched case–control study was conducted among 104 RT-PCR-confirmed COVID-19 patients recruited from two major hospitals designated as COVID-19 treating centres and 104 RT-PCR negative adults recruited from the catchment area of the two hospitals. Non-fasting blood samples obtained from cases upon hospital admission and from controls upon release from 14-day quarantine period were analysed for serum 25(OH)D using chemiluminescence assay. Serum level < 50.0 nmol/L was identified as vitamin D deficiency (VDD). Results: Cases were older while the controls had fewer persons with co-morbidities. Cases (median = 30.0; IQR = 21.2 nmol/L) had significantly lower 25(OH)D compared to controls (median = 35.9; IQR = 28.3 nmol/L) (p = 0.02), which persisted after adjustments (p = 0.026), along with Sinhalese ethnicity (p < 0.001). VDD was significantly more prevalent in cases (83.7% vs. 71.2%; crude odds ratio (OR) = 2.1; 95% CI 1.1, 4.1); but was not an independent predictor of COVID-19 status (adjusted OR = 1.9; 95% CI 0.6, 5.7). A lower 25(OH)D level was observed in moderate/severe cases (median = 28.0; IQR = 20.4 nmol/L) vs. mild (median = 39.8; IQR = 19.0 nmol/L), however, neither low serum concentrations nor deficiency showed an independent relationship with severity (p > 0.05). Diabetes was the sole predictor of COVID-19 severity (p = 0.022). Conclusions: Vitamin D has potential as a primary preventive strategy in preventing COVID-19 like infections, but not as a secondary preventive strategy to reduce its severity. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of extremely low-frequency electromagnetic fields (ELF-EMF) on myogenesis during the early stage of denervated muscle atrophy in Sprague-Dawley (SD) rats. Methods: a total of 36 female SD rats were fed in cages and divided into three groups: the normal control group, the muscle atrophy group and the 10Hz/1.5mT magnetic field group. The rats in the 10Hz/1.5mT magnetic field group received a magnetic field intervention for 2h per day. The posterior muscle groups of the lower limbs of rats were sampled on the first, second, third and fourth weeks for evaluating the underlying changes. The harvested tissues were stained with hematoxylin–eosin (HE) dye to observe the morphology of the muscle atrophy and to measure the muscle fiber cross-sectional area ratio. A reverse transcription polymerase chain reaction was used to detect the expression of Myf5 mRNA in each group. The myogenesis-related proteins in the harvested muscle tissue were detected by western blot assay. Results: (1) HE staining showed that with the extension of denervation time, the structural disorder of muscle fibers was aggravated, and in the second week of the denervated muscle atrophy, muscle fiber cross-sectional area tended to increase in the 10Hz/1.5mT magnetic field group. Overall muscle atrophy was inevitable. (2) Results of RT-PCR showed that the expression of Myf5 mRNA in the 10Hz/1.5mT magnetic field group was significantly high in the second week of denervation, and the difference was statistically significant compared with the muscle atrophy group (*p<0.05). (3) Western blot analysis showed that the expression of MYF5 proteins in the 10Hz/1.5mT magnetic field group was significantly higher than that in the muscle atrophy group (**p<0.01) in the second week of denervation. Further, the expression of MYOG protein in the 10Hz/1.5mT magnetic field group was significantly higher than that in the muscle atrophy group (****p<0.0001). Conclusions: 10Hz/1.5mT ELF-EMF can significantly promote myogenesis during early denervated muscle atrophy in SD rats. [ABSTRACT FROM AUTHOR]

The worldwide COVID-19 pandemic has changed people's lives and the diagnostic landscape. The nucleic acid amplification test (NAT) as the gold standard for SARS-CoV-2 detection has been applied in containing its transmission. However, there remains a lack of an affordable on-site detection system at resource-limited areas. In this study, a low cost "sample-in-answer-out" system incorporating nucleic acid extraction, purification, and amplification was developed on a single macrochannel-to-digital microfluidic chip. The macrochannel fluidic subsystem worked as a world-to-chip interface receiving 500–1000 μL raw samples, which then underwent bead-based extraction and purification processes before being delivered to DMF. Electrodes actuate an eluent dispensed to eight independent droplets for reverse transcription quantitative polymerase chain reaction (RT-qPCR). By reading with 4 florescence channels, the system can accommodate a maximum of 32 detection targets. To evaluate the proposed platform, a comprehensive assessment was conducted on the microfluidic chip as well as its functional components (i.e., extraction and amplification). The platform demonstrated a superior performance. In particular, using clinical specimens, the chip targeting SARS-CoV-2 and Flu A/B exhibited 100% agreement with off-chip diagnoses. Furthermore, the fabrication of chips is ready for scaled-up manufacturing and they are cost-effective for disposable use since they are assembled using a printed circuit board (PCB) and prefabricated blocks. Overall, the macrochannel-to-digital microfluidic platform coincides with the requirements of point-of-care testing (POCT) because of its advantages: low-cost, ease of use, comparable sensitivity and specificity, and availability for mass production. [ABSTRACT FROM AUTHOR]

Brain organoids have become a valuable tool for studying human brain development, disease modeling, and drug testing. However, generating brain organoids with mature neurons is time-intensive and often incomplete, limiting their utility in studying age-related neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report the generation of 3D brain organoids from human fibroblasts through direct reprogramming, with simplicity, efficiency, and reduced variability. We also demonstrate that induced brain organoids from APOE ε4 AD patient fibroblasts capture some disease-specific features and pathologies associated with APOE ε4 AD. Moreover, APOE ε4-induced brain organoids with mutant APP overexpression faithfully recapitulate the acceleration of AD-related pathologies, providing a more physiologically relevant and patient-specific model of familial AD. Importantly, transcriptome analysis reveals that gene sets specific to APOE ε4 patient-induced brain organoids are highly similar to those of APOE ε4 post-mortem AD brains. Overall, induced brain organoids from direct reprogramming offer a promising approach for more efficient and controlled studies of neurodegenerative disease modeling. [ABSTRACT FROM AUTHOR]

Effective bioremediation of a phenol-polluted environment harnesses microorganisms' ability to utilize hazardous compounds as beneficial degraders. In the present study, a consortium consisting of 15 bacterial strains was utilized. The current study aims to monitor the phenol biodegradation pathway. The tested consortium showed effective potential in the bioremediation of phenolcontaminated industrial wastewater. The enzymatic studies conducted brought to light that the bacterial consortium under test was proficient in degrading phenol under aerobic conditions while exhibiting the simultaneous expression of both ortho- and meta-cleavage pathways. It was observed that pheA, pheB, and C12O genes were maximally expressed, and the enzymes responsible for phenol degradation, namely, phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase, reached maximum activity after 48 h of incubation with a 20-ppm phenol concentration. To gain a deeper understanding of the activation of both ortho- and meta-cleavage pathways involved in phenol degradation, a technique known as differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was applied. This method allowed for the specific amplification and detection of genes responsible for phenol degradation. The expression levels of these genes determined the extent to which both ortho- and meta-cleavage pathways were activated in response to the presence of phenol. [ABSTRACT FROM AUTHOR]

Objective Materials and Methods Results Conclusion To investigate Treponema pallidum detection using immunohistochemistry (IHC) and reverse transcription‐polymerase chain reaction (RT‐PCR) assays in acquired oral syphilis (AOS).Thirty‐seven paraffin‐embedded tissue specimens of AOS (32 secondary and five primary) were analyzed, integrating double‐positive serological results with clinicodemographic and histopathological data. T. pallidum presence was semiquantitatively assessed by IHC, while RT‐PCR targeted T. pallidum DNA. Sensitivity, specificity, and the area under the curve (AUC) were calculated with 95% confidence intervals (CI).The study included mostly females (62.2%) with a mean age of 27.1 years. T. pallidum was detected in all samples by IHC, predominantly in the epithelium across all layers (43.2%). RT‐PCR identified T. pallidum DNA in 32 cases, with negative results observed in cases of secondary AOS. The AUC for IHC versus disease stage was 62.5% (95% CI: 45.1–77.8), and for RT‐PCR, it was 57.8% (95% CI: 40.5–73.8). The AUC comparing IHC to RT‐PCR was 83.8% (95% CI: 67.9–93.8).This study represents the first attempt to evaluate the proposed direct detection algorithm for AOS. IHC and RT‐PCR serve as ancillary tools for detecting T. pallidum in both primary and secondary stages of AOS. [ABSTRACT FROM AUTHOR]