Real-time PCR Assay Research Articles

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, Brachyspira hyodysenteriae, and Lawsonia intracellularis.

PEDV, Brachyspira hyodysenteriae, and Lawsonia intracellularis, are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens. Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of B. hyodysenteriae, and the 16S rDNA gene of L. intracellularis, specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/μL for B. hyodysenteriae and L. intracellularis, and 100 copies/μL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, B. hyodysenteriae and L. intracellularis. Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for B. hyodysenteriae, and 98.6% for L. intracellularis. The co-infection rates were 16.85% for PEDV + B. hyodysenteriae, 31.46% for PEDV + L. intracellularis, 57.86% for B. hyodysenteriae + L. intracellularis, and 16.85% for PEDV + B. hyodysenteriae + L. intracellularis, respectively. The new multiplex real-time PCR method can simultaneously differentiate PEDV, B. hyodysenteriae and L. intracellularis, making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.

Rutin Ameliorates Inflammation and Oxidative Stress in Ulcerative Colitis by Inhibiting NLRP3 Inflammasome Signaling Pathway.

Ulcerative colitis (UC) is an idiopathic inflammatory disease. We intend to explore the mechanism of Rutin in the therapy of UC. Disease activity index (DAI) and hematoxylin-eosin staining were employed to assess therapeutic effect of Rutin on dextran sulfate sodium-stimulated mice. The proliferation was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Oxidative stress (OS) was assessed by measuring reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Inflammatory factors were detected using enzyme-linked immunosorbent assay and immunofluorescence staining. mRNA and protein expressions were detected by real-time quantitative polymerase chain reaction and immunoblotting assay. Rutin decreased DAI scores and ameliorated pathological damage in UC mice with decreased levels of inflammatory factors. Rutin recovered the inhibited proliferation of fetal human colon cells caused by lipopolysaccharide. Rutin inhibited OS by reducing ROS and MDA, while enhancing SOD activity in LPS-induced fetal human colon cells. Rutin inhibited NLRP3 inflammasome in UC mice and cell model. Silencing NLRP3 enhanced the inhibitory effect of Rutin on OS in lipopolysaccharide-induced fetal human colon cells. Conversely, NLRP3 overexpression reversed the restraining role of Rutin in OS. Rutin ameliorates UC by inhibiting inflammation and OS through suppressing NLRP3 inflammasome.

Bone Marrow Mesenchymal Stem Cells-Derived Extracellular Vesicle miR-208a-3p Alleviating Spinal Cord Injury via Regulating the Biological Function of Spinal Cord Neurons.

We aim to explore the potential mechanism of bone marrow mesenchymal stem cells-derived extracellular vesicles (BMSCs-Exo) in improving spinal cord injury (SCI). Thirty male 12-week specific pathogen-free (SPF) Sprague-Dawley (SD) rats were used to construct SCI model invivo. Ten male 12-week SPF SD rats were used to extract BMSCs. The Basso, Beattie, Bresnahan (BBB) score was used to evaluate the motor function of rats. Real-time fluorescence quantitative PCR (RT-PCR), western blot (WB), and double luciferase assay were used to explore the regulation between rno-miR-208a-3p and Cdkn1a (p21) in BMSCs. Primary spinal cord neurons were treated with lipopolysaccharide (100 ng/mL) for 30 min to mimic SCI in vitro. Compared with the model group (14 scores), BMSCs-Exo increased BBB score (19 scores) in SCI rats. Compared with the sham group, Cdkn1a was upregulated, whereas rno-miR-208a-3p was downregulated in the model group. However, compared with the model group, Cdkn1a was downregulated, whereas rno-miR-208a-3p was upregulated in the BMSCs-Exo group. In addition, rno-miR-208a-3p inhibited the expression of Cdkn1a via direct binding way. BMSCs-Exo-rno-miR-208a-3p promoted the proliferation of primary spinal neurons via inhibiting apoptosis in vitro. Moreover, BMSCs-Exo-rno-miR-208a-3p promoted cyclin D1, CDK6, and Bcl-2 and inhibited Bax expression in a cell model of SCI. In conclusion, BMSCs-Exo-carried rno-miR-208a-3p significantly protects rats from SCI via regulating the Cdkn1a pathway.

Longitudinal tracing of on-treatment plasma Epstein-Barr virus DNA as a biomarker for real-time dynamic risk monitoring in patients with nasopharyngeal carcinoma: The EP-SEASON study.

136 Background: Plasma Epstein-Barr virus (EBV) DNA is a biomarker for nasopharyngeal carcinoma (NPC). However, whether longitudinal on-treatment EBV DNA tracing would inform real-time recurrence risk in NPC remains to be elucidated. To address this issue, we conducted EP-SEASON (NCT03855020), a prospective cohort study of longitudinal on-treatment EBV DNA in EBV-positive NPC patients. Methods: Non-metastatic NPC with detectable pretreatment EBV DNA that received standard sequential chemo-radiotherapy were recruited. Serial EBV DNA in blood specimens were collected across pretreatment, 3-week after each induction chemotherapy (IC), every-week during radiotherapy (RT), within 1-week and 1-3 months upon RT completion. EBV DNA extracted from plasma was quantified by a real-time quantitative polymerase chain reaction assay targeting the BamHI-W region of the EBV genome. Longitudinal changing patterns of EBV DNA and their clinical relevance were analyzed. Results: Between May 2019 and April 2021, a total of 1000 patients underwent per-protocol EBV DNA assessments and treatment; 248 and 752 patients received concurrent chemoradiotherapy (CCRT, subcochortno-IC) and IC plus CCRT (subcochortIC), respectively. Longitudinal changes and clearance rates of EBV DNA displayed unique patterns during IC and RT phases. Throughout IC and RT phases, the rate of EBV DNA clearance decreased with repeated IC cycles; interestingly, the rate re-rose upon the introduction of RT, and then dropped to low levels (<10%/week) after the 4th week of RT (post-RT4w). Patients in subcochortno-IC and subcochortIC also exhibited differential clearance patterns during RT, with the rate of clearance being higher in subcochortno-IC before the 2nd week of RT (post-RT2w). Notably, despite the prognostic significance of EBV DNA at each timepoint ( Pall < 0.05), longitudinal EBV DNA displayed a linkage changing map with dynamic recurrence risks: 3-year disease-free survival (DFS) rate steadily dropped with delayed EBV DNA clearance during IC; it re-rose at the 2nd RT week upon EBV DNA clearance, followed by a sharp decrease at the 4th RT week in subcochortIC. In contrast, 3-year DFS rate continuously decreased with delayed EBV DNA clearance in subcochortno-IC. Finally, we identified different phenotypes of whole-course circulating tumor DNA changing dynamics that showed distinct prognosis ( P < 0.05). Conclusions: Longitudinal on-treatment EBV DNA tracing could forecast real-time risks and facilitate risk-adapted decision-making in NPC.

Validation of real-time PCR assays for detecting Plasmodium and Babesia DNA species in blood samples

Malaria and babesiosis are global health threats affecting humans, wildlife, and domestic animals, particularly in Africa, the Americas, and Europe. Malaria can lead to severe outcomes, while babesiosis usually resembles a mild illness but can be severe and fatal in individuals with weakened immune systems. Swift, accurate detection of these parasites is crucial for treatment and control. We evaluated a real-time PCR assay for diagnosing five Plasmodium and three Babesia species from blood samples, assessing its sensitivity, specificity, and analytical performance by analyzing 46 malaria-positive and 32 Babesia spp-positive samples diagnosed through microscopy. The limit of detection for Plasmodium species ranged from 30 to 0.0003 copies/µL. For mixed infections, it was 0.3 copies/µL for P. falciparum/P. vivax and 3 copies/µL for P. malariae/P. knowlesi. Babesia species had a detection limit of 0.2 copies/µL. No cross-reactivity was observed among 64 DNA samples from various microorganisms. The assay showed good sensitivity, detecting Plasmodium and Babesia species with 100 % accuracy overall, except for P. falciparum (97.7 %) and B. microti (12.5 %). The low sensitivity of detecting B. microti was attributed to limitations in microscopy for species identification. This technique heavily relies on the proficiency of the examiner, as species within the genus cannot be distinguished under a microscope. Additionally, Babesia can be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. The findings support multiplex qPCR's diagnostic superiority over the gold standard, despite higher costs. It offers enhanced sensitivity, specificity, and detects mixed infections, crucial for effective monitoring and diagnosis of malaria and babesiosis in endemic regions with significant public health challenges.

Burden and epidemiology of Campylobacter species in acute enteritis cases in Burkina Faso

BackgroundCampylobacter spp. is a significant etiological agent of bacterial gastroenteritis globally. In Burkina Faso (BFA), the actual impact of this pathogen on gastroenteritis is considerably underestimated, primarily due to inadequate surveillance systems.ObjectivesThis study aimed to investigate the proportion of Campylobacter species responsible for acute gastroenteritis among patients of all ages in urban and rural areas of BFA, using molecular biology techniques.Study design & methodsBetween 2018 and 2021, faecal specimens were obtained from 1,295 individuals presenting with acute gastroenteritis. These samples underwent screening for the Campylobacter coli/jejuni/lari complex utilizing real-time polymerase chain reaction (PCR) assays. Subsequently, positive samples were subjected to species-level differentiation through the application of species-specific primers.ResultsCampylobacter spp. was detected in 25.0% (324/1,295) of the samples analysed. The majority of positive samples (95%, 308/324) were obtained from children under 5 years of age. Species identification was performed on a subset of 114 isolates, revealing 51 Campylobacter jejuni, 10 Campylobacter coli, and 53 Campylobacter isolates that remained unspeciated.ConclusionsThis study reveals a significant prevalence of Campylobacter species among patients with acute gastroenteritis, with a particularly high incidence observed in children under 5 years of age. Based on these findings, the implementation of routine Campylobacter surveillance in public health laboratories is strongly recommended to better monitor and address this health concern.

Hsa-miR-181a-2-3p inhibits the oncogenicity of colon cancer by directly targeting STING.

Colon cancer is a common malignant tumor of the gastrointestinal system, which is characterized by high morbidity and mortality. The purpose of this study was to analyze the expression and biological role of miR-181a-2-3p in colon cancer and to investigate the molecular mechanism of its regulatory effect on colon cancer through stimulator of interferon genes (STING). Real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was used to detect the expression of miR-181a-2-3p in colon cancer cell lines and normal intestinal epithelial cells. After overexpression of miR-181a-2-3p in colon cancer cell lines SW480 and HT29, cells were examined by CCK8, Transwell, and flow cytometry assays for alterations in proliferation, migration, apoptosis, and cell cycle. Target genes of miR-181a-2-3p were predicted by bioinformatics and validated by dual luciferase assays. Rescue experiments were performed to explore the role of STING in the effect of miR-181a-2-3p. The effect of miR-181a-2-3p on colon cancer proliferation in vivo was validated by nude mouse tumorigenicity assay. miR-181a-2-3p was lowly expressed in both colon cancer tissues and cell lines. Overexpression of miR-181a-2-3p led to reduced proliferation and migration, increased apoptosis, and altered cell cycle in colon cancer cell lines SW480 and HT29. STING was a target gene of miR-181a-2-3p. Increased STING expression partially counteracted the effect of overexpression of miR-181a-2-3p on colon cancer cell lines. miR-181a-2-3p also suppressed colon cancer proliferation in vivo. miR-181a-2-3p inhibits the proliferation and oncogenicity of colon cancer, and its molecular mechanism could be inhibited by STING.

Real-time PCR assay to detect the novel Clade Ib monkeypox virus, September 2023 to May 2024.

Monkeypox virus (MPXV) is an emerging zoonotic pathogen with complex epidemiology necessitating rapid diagnosis and distinguishing between clades and subclades. The emerging Clade Ib lacks the genomic region used in the Clade I-specific assay from the Centers for Disease Control and Prevention. We report an MPXV real-time PCR to specifically detect Clade Ib. The assay demonstrated proficient sensitivity and specificity in 92 samples and can be included along other TaqMan-based assays to detect MPXV and distinguish between clades and subclades.

Longer ICU stay and invasive mechanical ventilation accelerate telomere shortening in COVID-19 patients 1 year after recovery

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes virus-induced-senescence. There is an association between shorter telomere length (TL) in coronavirus disease 2019 (COVID-19) patients and hospitalization, severity, or even death. However, it remains unknown whether virus-induced-senescence is reversible. We aim to evaluate the dynamics of TL in COVID-19 patients 1 year after recovery from intensive care units (ICU). Longitudinal study enrolling 49 patients admitted to ICU due to COVID-19 (August 2020 to April 2021). Relative telomere length (RTL) quantification was carried out in whole blood by monochromatic multiplex real-time quantitative PCR (MMqPCR) assay at hospitalization (baseline) and 1 year after discharge (1-year visit). The association between RTL and ICU length of stay (LOS), invasive mechanical ventilation (IMV), prone position, and pulmonary fibrosis development at 1-year visit was evaluated. The median age was 60 years, 71.4% were males, median ICU-LOS was 12 days, 73.5% required IMV, and 38.8% required a prone position. Patients with longer ICU-LOS or who required IMV showed greater RTL shortening during follow-up. Patients who required pronation had a greater RTL shortening during follow-up. IMV patients who developed pulmonary fibrosis showed greater RTL reduction and shorter RTL at the 1-year visit. Patients with longer ICU-LOS and those who required IMV had a shorter RTL in peripheral blood, as observed 1 year after hospital discharge. Additionally, patients who required IMV and developed pulmonary fibrosis had greater telomere shortening, showing shorter telomeres at the 1-year visit. These patients may be more prone to develop cellular senescence and lung-related complications; therefore, closer monitoring may be needed.

A rapid on-site loop-mediated isothermal amplification technology as an early warning system for the detection of Shiga toxin-producing Escherichia coli in water.

Shiga toxin-producing Escherichia coli (STEC) is an important waterborne pathogen capable of causing serious gastrointestinal infections with potentially fatal complications, including haemolytic-uremic syndrome. All STEC serogroups harbour genes that encode at least one Shiga toxin (stx1 and/or stx2), which constitute the primary virulence factors of STEC. Loop-mediated isothermal amplification (LAMP) enables rapid real-time pathogen detection with a high degree of specificity and sensitivity. The aim of this study was to develop and validate an on-site portable diagnostics workstation employing LAMP technology to permit rapid real-time STEC detection in environmental water samples. Water samples (n=28) were collected from groundwater wells (n=13), rivers (n=12), a turlough (n=2) and an agricultural drain (n=1) from the Corrib catchment in Galway. Water samples (100 ml) were passed through a 0.22 µm filter, and buffer was added to elute captured cells. Following filtration, eluates were tested directly using LAMP assays targeting stx1, stx2 and E. coli phoA genes. The portable diagnostics workstation was used in field studies to demonstrate the on-site testing capabilities of the instrument. Real-time PCR assays targeting stx1 and stx2 genes were used to confirm the results. The limit of detection for stx1, stx2 and phoA LAMP assays were 2, 2 and 6 copies, respectively. Overall, stx1, stx2 and phoA genes were detected by LAMP in 15/28 (53.6 %), 9/28 (32.2 %) and 24/28 (85.7 %) samples, respectively. For confirmation, the LAMP results for stx1 and stx2 correlated perfectly (100 %) with those obtained using PCR. The portable diagnostics workstation exhibited high sensitivity throughout the on-site operation, and the average time from sample collection to final result was 40 min. We describe a simple, transferable and efficient diagnostic technology for on-site molecular analysis of various water sources. This method allows on-site testing of drinking water, enabling evidence-based decision-making by public health and water management authorities.

Evaluation of the Fluorion HCV QNP v3.0 real-time PCR assay for quantifying HCV RNA in Moroccan patients: A comparative study with COBAS AmpliPrep/COBAS TaqMan HCV v2.0

PurposeHepatitis C Virus (HCV) RNA quantification is crucial for diagnosing and monitoring chronic HCV treatment. Cost-effective methods are crucial to ensure accessibility. This study evaluated the Fluorion HCV QNP v3.0 Real-Time PCR assay's effectiveness in EDTA-plasma and serum, comparing it with the COBAS AmpliPrep/COBAS TaqMan HCV v2.0 (CAP/CTM HCV v2.0) test. Methods105 matched pairs of EDTA-plasma and serum specimens (91 positive and 14 negative) from HCV infected Moroccan patients were analyzed using the Fluorion HCV QNP v3.0 assay and compared to the CAP/CTM HCV v2.0 test, being the reference method. ResultsThe values obtained by the Fluorion HCV QNP v3.0 in plasma were slightly higher than those in serum (3.94 ± 2.23 log10 IU/mL versus 3.91 ± 2.22 log10 IU/mL) and were both significantly lower than those quantified by the CAP/CTM HCV v2.0 assay (4.34 ± 2.28 log10 IU/mL; p < 0.001). High correlations were observed between the Fluorion HCV QNP v3.0 serum and CAP/CTM HCV v2.0 (R2 = 0.9433), the Fluorion HCV QNP v3.0 plasma and CAP/CTM HCV v2.0 (R2 = 0.949) and the Fluorion HCV QNP v3.0 serum and plasma (R2 = 0.9954). HCV RNA was detected in all tested genotypes by both assays. ConclusionThe Fluorion HCV QNP v3.0 assay demonstrated excellent performance in comparison with the CAP/CTM HCV v2.0 on both plasma and serum samples which can be used interchangeably for HCV quantification. The test was shown to be suitable for disease monitoring including all HCV genotypes.

Commiphora myrrha n-hexane extract suppressed breast cancer progression through induction of G0/G1 phase arrest and apoptotic cell death by inhibiting the Cyclin D1/CDK4-Rb signaling pathway.

Breast cancer (BC) is one of the most frequently observed malignancies globally, yet drug development for BC has been encountering escalating challenges. Commiphora myrrha is derived from the dried resin of C. myrrha (T. Nees) Engl., and is widely adopted in China for treating BC. However, the anti-BC effect and underlying mechanism of C. myrrha remain largely unclear. MTT assay, EdU assay, and colony formation were used to determine the effect of C. myrrha n-hexane extract (CMHE) on the proliferation of human BC cells. Cell cycle distribution and apoptosis were assessed via flow cytometry analysis. Moreover, metastatic potential was evaluated using wound-scratch assay and matrigel invasion assay. The 4T1 breast cancer-bearing mouse model was established to evaluate the anti-BC efficacy of CMHE in vivo. RNA-sequencing analysis, quantitative real-time PCR, immunoblotting, immunohistochemical analysis, RNA interference assay, and database analysis were conducted to uncover the underlying mechanism of the anti-BC effect of CMHE. We demonstrated the significant inhibition in the proliferative capability of BC cell lines MDA-MB-231 and MCF-7 by CMHE. Moreover, CMHE-induced G0/G1 phase arrest and apoptosis of the above two BC cell lines were also observed. CMHE dramatically repressed the metastatic potential of these two cells in vitro. Additionally, the administration of CMHE remarkably suppressed tumor growth in 4T1 tumor-bearing mice. No obvious toxic or side effects of CMHE administration in mice were noted. Furthermore, immunohistochemical (IHC) analysis demonstrated that CMHE treatment inhibited the proliferative and metastatic abilities of cancer cells, while also promoting apoptosis in the tumor tissues of mice. Based on RNA sequencing analysis, quantitative real-time PCR, immunoblotting, and IHC assay, the administration of CMHE downregulated Cyclin D1/CDK4-Rb signaling pathway in BC. Furthermore, RNA interference assay and database analysis showed that downregulated Cyclin D1/CDK4 signaling cascade participated in the anti-BC activity of CMHE. CMHE treatment resulted in the suppression of BC cell growth through the stimulation of cell cycle arrest at the G0/G1 phase and the induction of apoptotic cell death via the inhibition of the Cyclin D1/CDK4-Rb pathway, thereby enhancing the anti-BC effect of CMHE. CMHE has potential anti-BC effects, particularly in those harboring aberrant activation of Cyclin D1/CDK4-Rb signaling.

Analytical and clinical evaluation of a novel real-time PCR-based detection kit for Mpox virus

Outbreaks of emerging diseases, like Mpox in 2022, pose unprecedented challenges to global healthcare systems. Although Mpox cases globally decreased since the end of 2022, numbers are still significant in the African Region, European Region, Region of the Americas, and Western Pacific Region. Rapid and efficient detection of infected individuals by precise screening assays is crucial for successful containment. In these assays, analytical and clinical performance must be assessed to ensure high quality. However, clinical studies evaluating Mpox virus (MPXV) detection kits using patient-derived samples are scarce. This study evaluated the analytical and clinical performance of a new diagnostic MPXV real-time PCR detection kit (Sansure Monkeypox Virus Nucleic Acid Diagnostic Kit) using patient-derived samples collected in Germany during the MPXV clade IIb outbreak in 2022. Our experimental approach determined the Limit of Detection (LoD) to less than 200 cp/mL using whole blood samples and samples derived from vesicles or pustules. Furthermore, we tested potentially inhibiting substances and pathogens with homologous nucleic acid sequences or similar clinical presentation and detected no cross-reactivity or interference. Following this, the assay was compared to a CE-marked test in a clinical performance study and achieved a diagnostic sensitivity of 100.00% and diagnostic specificity of 96.97%. In summary, the investigated real-time PCR assay demonstrates high analytical performance and concurs with the competitor device with high specificity and sensitivity.

Utilization of multiplex polymerase chain reaction for simultaneous and rapid detection of viral infections from different ocular structures

The impact of viral keratitis (VK) on individuals and society is notable. Early diagnosis and treatment are crucial in managing viral keratitis effectively. Timely intervention with antiviral medications and supportive care can help mitigate the severity of the infection and improve visual outcomes. We examined the prevalence of varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), adenovirus (AdV) and herpes simplex virus type 2 (HSV-2) in patients suspected for ocular infections. Patients included in the study exhibited various clinical manifestations indicative of ocular pathology, such as infectious keratitis, corneal scar, endogenous endophthalmitis, panuveitis, endothelitis, stromal edema, and other relevant conditions. Four different types of tear fluid, corneal samples epithelium, aqueous humor and vitreous humor were taken. After genome extraction, multiplex real-time PCR was used for diagnosis of viruses. 48 (29.6%) out of the total of 162 (100%) eye specimen were positive. The dominant prevalence was VZV (12.3%) and HSV-1 (11.7%) followed by AdV (4.9%) and HSV-2 (0.6%). There were 4 (8.3%) coinfections within the samples (HSV-1 and VZV). Aqueous humor samples demonstrated superior virus detection ability and our only HSV-2 positive sample was from aqueous humor. The utilization of multiplex real-time PCR assays in differential diagnosis of VK holds promise for expeditious diagnoses while also preventing unwarranted antibiotic prescriptions. Moreover, the aqueous humor appears to be a more sensitive site for detecting viral keratitis.

The preventive and therapeutic role of Lactobacillus spp. in in vitro model of inflammation via affecting autophagy signaling pathway.

Intestinal inflammation has various causes and leads to some inflammatory diseases, of which autophagy pathway dysfunction could be considered as one of them. Probiotics could have a positive effect on reducing inflammation by activating the autophagy pathway. To evaluate the precise effects of probiotics as preventive and therapeutic agents to control the symptoms of inflammatory diseases, we aimed to investigate the efficacy of Lactobacillus spp. in regulating the autophagy signaling pathway. A quantitative real-time polymerase chain reaction assay was used to analyze the expression of autophagy genes involved in the formation of phagophores, autophagosomes, and autolysosomes after exposing the HT-29 cell line to sonicated pathogens and adding Lactobacillus spp. before, after, and simultaneously with inflammation. A cytokine assay was also accomplished to evaluate the interleukin(IL)-6 and IL-1β level following the probiotic treatment. Lactobacillus spp. generally increased autophagy gene expression and consumption of Lactobacillus spp. before, simultaneously, and after inflammation, ultimately leading to activate autophagy pathways. The proinflammatory cytokines including IL-6and IL-1β decreased after probiotic treatment. Our native probiotic Lactobacillus spp. showed beneficial effects on HT-29 cells by increasing autophagy gene expression and decreasing the proinflammatory cytokines production in all treatments. Therefore, this novel probiotic cocktail Lactobacillus spp. can prevent and treat inflammation-related diseases.

Predictive biomarkers for metachronous gastric cancer development after endoscopic resection of early gastric cancer.

We aimed to identify predictive markers for metachronous gastric cancer (MGC) in early gastric cancer (EGC) patients curatively treated with endoscopic submucosal dissection (ESD). From EGC patients who underwent ESD, bulk RNA sequencing was performed on non-cancerous gastric mucosa samples at the time of initial EGC diagnosis. This included 23 patients who developed MGC, and 23 control patients without additional gastric neoplasms for over 3 years (1:1 matched by age, sex, and Helicobacter pylori infection state). Candidate differentially-expressed genes were identified, from which biomarkers were selected using real-time quantitative polymerase chain reaction and cell viability assays using gastric cell lines. An independent validation cohort of 55 MGC patients and 125 controls was used for marker validation. We also examined the severity of gastric intestinal metaplasia, a known premalignant condition, at initial diagnosis. From the discovery cohort, 86 candidate genes were identified of which KDF1 and CDK1 were selected as markers for MGC, which were confirmed in the validation cohort. CERB5 and AKT2 isoform were identified as markers related to intestinal metaplasia and were also highly expressed in MGC patients compared to controls (p<0.01). Combining these markers with clinical data (age, sex, H. pylori and severity of intestinal metaplasia) yielded an area under the curve (AUC) of 0.91 (95% CI, 0.85-0.97) for MGC prediction. Assessing biomarkers in non-cancerous gastric mucosa may be a useful method for predicting MGC in EGC patients and identifying patients with a higher risk of developing MGC, who can benefit from rigorous surveillance.

METTL3/IGF2BP1 influences the development of non-small-cell lung cancer by mediating m6A methylation modification of TRPV1.

Methyltransferase 3 (METTL3) accelerates N6-methyladenosine (m6A) modifications and affects cancer progression, including non-small-cell lung cancer (NSCLC). In this study, we aimed to explore the regulatory mechanisms of METTL3 underling NSCLC. Immunohistochemical assay, quantitative real-time polymerase chain reaction (qRT-PCR)assay, and western blot assay were conducted for gene expression. MTT assay and colony formation assay were performed to explore cell proliferation capacity. Cell apoptosis and THP-1 cell polarization were estimated by flow cytometry analysis. Cell migration and invasion capacities were evaluated by transwell assay. Methylated RNA immunoprecipitation assay, dual-luciferase reporter assay, actinomycin D treatment and RIP assay were performed to analyze the relationships of METTL3, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), and transient receptor potential cation channel subfamily V member 1 (TRPV1). The functions of METTL3 and TRPV1 in vivo were investigated through establishing the murine xenograft model. TRPV1 expression was upregulated in NSCLC and related poor prognosis. TRPV1 silencing inhibited NSCLC cell growth and metastasis, induced NSCLC cell apoptosis, and repressed M2 macrophage polarization. The results showed that METTL3 and IGF2BP1 could regulate TRPV1 expression through m6A methylation modification. Moreover, METTL3 deficiency inhibited NSCLC cell growth, metastasis, and M2 macrophage polarization and facilitated NSCLC cell apoptosis, while TRPV1 overexpression restored the impacts. In addition, METTL3 knockdown restrained tumor growth in vivo via regulating TRPV1 expression. METTL3 bound to IGF2BP1 and enhanced IGF2BP1's m6A recognition of TRPV1 mRNA, thereby promoting NSCLC cell growth and metastasis, and inhibiting M2 macrophage polarization.

PUS7-dependent pseudouridylation of ALKBH3 mRNA inhibits gastric cancer progression.

RNA pseudouridylation is a critical post-transcriptional modification that influences gene expression and impacts various biological functions. Despite its significance, the role of mRNA pseudouridylation in cancer remains poorly understood. This study investigates the impact of pseudouridine synthase 7 (PUS7)-mediated pseudouridylation of Alpha-ketoglutarate-dependent Dioxygenase alkB Homolog 3 (ALKBH3) mRNA in gastric cancer. Immunohistochemistry and Western blotting were used to assess PUS7 protein levels in human gastric cancer tissues. The relationship between PUS7 and gastric cancer progression was examined using 3D colony formation assays and subcutaneous xenograft models. Real-time quantitative PCR (RT-qPCR), Western blotting, and polysome profiling assays were conducted to investigate how PUS7 regulates ALKBH3. A locus-specific pseudouridine (Ψ) detection assay was used to identify Ψ sites on ALKBH3 mRNA. Our findings indicate a significant reduction of PUS7 in gastric cancer tissues compared to adjacent non-tumour tissues. Functional analyses reveal that PUS7 inhibits gastric cancer cell proliferation and tumour growth via its catalytic activity. Additionally, PUS7 enhances the translation efficiency of ALKBH3 mRNA by modifying the U696 site with pseudouridine, thereby attenuating tumour growth. Importantly, ALKBH3 functions as a tumour suppressor in gastric cancer, with its expression closely correlated with PUS7 levels in tumour tissues. PUS7-dependent pseudouridylation of ALKBH3 mRNA enhances its translation, thereby suppressing gastric cancer progression. These findings highlight the potential significance of mRNA pseudouridylation in cancer biology and suggest a therapeutic target for gastric cancer. PUS7 enhances the translation efficiency of ALKBH3 through its pseudouridylation activity on ALKBH3 mRNA, thereby inhibiting gastric tumourigenesis. The expression levels of PUS7 and ALKBH3 are significantly correlated in gastric tumours, which may be potential prognostic predictors and therapeutic targets for patients with gastric cancer.

An Improved Bulk DNA Extraction Method for Detection of Helicoverpa armigera (Lepidoptera: Noctuidae) Using Real-Time PCR.

Helicoverpa armigera is among the most problematic agricultural pests worldwide due to its polyphagy and ability to evolve pesticide resistance. Molecular detection methods for H. armigera have been developed to track its spread, as such methods allow for rapid and accurate differentiation from the native sibling species H. zea. Droplet digital PCR (ddPCR) is a preferred method for bulk screening due to its accuracy and tolerance to PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield, purity, and throughput are crucial for real-time PCR assay optimization. Bulk DNA extractions have recently been improved to where real-time PCR sensitivity can equal that of ddPCR, but these new methods require significant time and specialized equipment. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in lower Cq values during real-time PCR while reducing the processing time and cost per specimen. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of H. armigera.

Distribution and seasonal fluctuation of visceral leishmaniasis vectors sandflie in Lüliang City of Shanxi Province in 2023

To investigate the distribution and seasonal fluctuations of visceral leishmaniasis vectors sandflies in Lüliang City, Shanxi Province, so as to provide insights into assessment of the visceral leishmaniasis transmission risk and formulation of visceral leishmaniasis control measures. A total of 12 natural villages were sampled from Shilou County, Lishi District, Lanxian County, Linxian County and Wenshui County in Lüliang City, Shanxi Province from June to September, 2023, and sandflies were captured using light traps from 7 breeding habitats, including farmers' houses, sheep pens, cattle pens, chicken coops, pig pens, mule and horse pens, and loess-cave dwellings. Following morphological identification of the sandfly species, the distribution of sandflies and the seasonal fluctuations of the sandfly density were analyzed. In addition, the Leishmania was detected in sandflies using a real-time fluorescence quantitative PCR assay. A total of 2 831 sandflies were captured with 156 light traps in Lüliang City from June to September, 2023, including 2 638 female sandflies (93.18%) and 193 male sandflies (6.82%), and the average density was 16.91 sandflies/(light-night). The seasonal fluctuations of the sandfly density all appeared a unimodal distribution in all survey sites, and the sandfly density peaked in July and then declined rapidly. Among all types of breeding habitats, the greatest sandfly density was found in sheep pens [39.04 sandflies/(light-night)]. In addition, 4.08% (2/49) of the sandfly samples were tested positive for Leishmania nucleic acid as revealed by the real-time fluorescence quantitative PCR assay. Sandflies were widely distributed in Lüliang City, Shanxi Province in 2023, and the peak of the sandfly density was observed in July, which had a visceral leishmaniasis transmission risk. Intensified surveillance of visceral leishmaniasis and sandfly vectors is required and targeted vector control is recommended.