A novel quantitative real-time PCR with the GAPDH reference gene for peste des petits ruminants.

Peste des petits ruminants (PPR) is a serious acute, highly contagious disease caused by the peste des petits ruminants virus (PPRV). This study aims to establish a qRT-PCR assay with an internal amplification control for the rapid and accurate detection of PPRV. The primers and probes for PPRV N were based on the national standard of the diagnostic techniques for PPR of China, and a pair of primers and TaqMan probes for the internal reference gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was designed. Optimisation of the reaction conditions, specificity, sensitivity and reproducibility tests, and clinical sample detection were conducted. The results showed that the optimal primers and probe concentrations of PPRV were 0.4 µmol/l and 0.4 µmol/l, respectively, and were 0.4 µmol/l and 0.2 µmol/l for the reference gene GAPDH, respectively. The established method has no cross-reaction with other viruses. The minimum detection limit was 6.8 copies/µl for PPRV and 190 copies/µl for GAPDH. The coefficients of variation (CV%) of PPRV and GAPDH were both lower than 2%. The results suggest that the PPRV qRT-PCR method containing internal reference genes has strong specificity, high sensitivity, and good reproducibility. The addition of internal reference genes for the sample quality control improves the accuracy of the detection.

Development of a Multiplex Real-Time Quantitative PCR Assay for Detecting Vaginal Microbiota in Chinese Women - China, 2021-2022.

Introduction: The Nugent score, limited by subjectivity and personnel requirements, lacks accuracy. Establishing a precise and simple molecular test is therefore essential for detecting vaginal microbiota compositions and evaluating vaginal health. Methods: We evaluated the vaginal health of Chinese women using quantitative polymerase chain reaction (qPCR) to target Lactobacillus crispatus (L. crispatus), L. iners, Gardnerella vaginalis (G. vaginalis), Atopobium vaginae (A. vaginae), and Megasphaera phylotype1. bacterial vaginosis (BV)-related bacteria shared a fluorescent channel. Using 16S rDNA sequencing as a reference standard, we evaluated and validated the diagnostic accuracy of the qPCR assay. Results: Both qPCR and 16S rDNA sequencing demonstrated 90.5% concordance in segregating vaginal community state type (CST), as visualized through heatmaps and PCoA. Spearman's correlation analysis revealed strong correlations between the two methods in calculating the RA of L. crispatus (CST I), L. iners (CST III), and BV-related bacteria (CST IV), with coefficients of 0.865, 0.837, and 0.827, respectively. Receiver operating characteristic analysis showed that qPCR had significant diagnostic accuracy for CST I, CST III, and CST IV (molecular BV), with area under the curve values of 0.967, 0.815, and 0.950, respectively, indicating strong predictive power. Discussions: Vaginal health can be evaluated using a single qPCR amplification experiment, making the multiplex qPCR assay a highly accurate tool for this purpose.

Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring ß-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through ß-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Development and evaluation of a rapid diagnostic method for Sporothrix globosa in Asia using quantitative real-time PCR.

BACKGROUND: Sporotrichosis is a chronic granulomatous infection of the skin and subcutaneous tissue that can affect any organ through lymphatic spread. The prevalence of sporotrichosis infections is increasing and its treatment is challenging as there are no unified and standard diagnostic techniques or antifungal medications. Controlling further spread requires a rapid diagnosis. Assessment of clinical symptoms, histological analysis, serological testing, and pathogen culture are all necessary for the diagnosis of sporotrichosis. However, these procedures are unable to identify the species. The development of safe, reliable, and species-specific diagnostic techniques is essential. OBJECTIVE: To establish and evaluate a new quantitative real-time PCR assay for the rapid diagnosis of sporotrichosis and to identify relevant species. METHODS: Polymorphisms in calmodulin (CAL) gene sequences and the internal transcribed spacer (ITS) were used in a quantitative real-time PCR assay to identify S. globosa, S. schenckii, and non-target species. RESULTS: The quantitative real-time PCR assay had 100% sensitivity and specificity. The limit of detection was 6 fg/µl. Thirty-four clinical specimens were verified to be infected with S. globosa with a 100% positive detection rate. CONCLUSIONS: The quantitative PCR technique developed in this study is a quick, accurate, and targeted method of identifying S. globosa based on polymorphisms in CAL sequences and ITS. It can be used for a prompt clinical diagnosis to identify S. globosa in clinical specimens from patients with sporotrichosis.

Utility of pan-bacterial and pan-fungal PCR in endophthalmitis: case report and review of the literature.

BACKGROUND: Endophthalmitis is a clinical diagnosis but identification of the disease-causing agent or agents allows for a more tailored treatment. This is routinely done through intraocular fluid cultures and staining. However, culture-negative endophthalmitis is a relatively common occurrence, and a causative organism cannot be identified. Thus, further diagnostic testing, such as pan-bacterial and pan-fungal polymerase chain reactions (PCRs), may be required. BODY: There are now newer, other testing modalities, specifically pan-bacterial and pan-fungal PCRs, that may allow ophthalmologists to isolate a causative agent when quantitative PCRs and cultures remain negative. We present a case report in which pan-fungal PCR was the only test, amongst quantitative PCRs, cultures, and biopsies, that was able to identify a pathogen in endophthalmitis. Pan-PCR has unique advantages over quantitative PCR in that it does not have a propensity for false-positive results due to contamination. Conversely, pan-PCR has drawbacks, including its inability to detect viruses and parasites and its increased turnaround time and cost. Based on two large retrospective studies, pan-PCR was determined not to be recommended in routine cases of systemic infection as it does not typically add value to the diagnostic workup and does not change the treatment course in most cases. However, in cases like the one presented, pan-bacterial and pan-fungal PCRs may be considered if empiric treatment fails or if the infective organism cannot be isolated. If pan-PCR remains negative or endophthalmitis continues to persist, an even newer form of testing, next-generation sequencing, may aid in the diagnostic workup of culture-negative endophthalmitis. CONCLUSION: Pan-bacterial and pan-fungal PCR testing is a relatively new diagnostic tool with unique advantages and drawbacks compared to traditional culturing and PCR methods. Similar to the tests' use in non-ophthalmic systemic infections, pan-bacterial and pan-fungal PCRs are unlikely to become the initial diagnosis test and completely replace culture methods. However, they can provide useful diagnostic information if an infectious agent is unable to be identified with traditional methods or if empiric treatment of endophthalmitis continues to fail.

Detection of Mycobacterium tuberculosis complex in saliva by quantitative PCR: A potential alternative specimen for pulmonary tuberculosis diagnosis.

BACKGROUND: Current tuberculosis (TB) diagnostic tests primarily rely on sputum samples, yet many TB patients cannot produce sputum. This study explored whether saliva could be used instead of sputum to diagnose pulmonary TB (PTB). METHOD: The study included 32 patients with confirmed PTB and 30 patients with other respiratory diseases (ORD). Saliva from all study participants was subjected to quantitative (qPCR) assays targeting the IS1081 gene for detection of M. tuberculosis complex DNA. RESULTS: The sensitivity of saliva IS1081 qPCR was 65.6 % (95 % CI 48.4-80.2 %) with positive results for 21/32 PTB cases, while the specificity was 96.7 % (95 % CI 85.9-99.6 %) with negative results for 29/30 participants with ORD. Sensitivity improved to 72.4 % (95 % CI 54.6-86.0 %) when sputum-Xpert was used as the reference standard, while remaining similar at 65.5 % (95 % CI 47.4-80.7 %) when culture was used as the reference standard. In receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) for saliva IS1081 qPCR was 82.5 % (95 % CI 71.7-93.3 %). CONCLUSION: Saliva testing offers a promising alternative to sputum for TB diagnosis among confirmed PTB cases. Larger multicenter studies, encompassing diverse clinical TB characteristics, are needed to provide improved estimates of diagnostic sensitivity and specificity.

Expression Levels and Clinical Values of miR-195, miR-424, miR-10b, miR-103a-3p, and miR-542-3p in Vasculo-Behçet's Disease.

Objective: MicroRNAs (miRNAs) are involved in a range of pathological and biological processes. Vascular involvement is an important complication associated with morbidity and mortality in Behçet's disease (BD). In this study, we aimed to evaluate the expression levels of miR-195, miR-424, miR-10b, miR-103a-3p, and miR-542-3p in Turkish patients with BD, and their possible association with vascular involvement and clinical activity. Methods: This cross-sectional study included 61 BD patients and 25 age- and sex-matched healthy individuals. The patients were categorised into two groups based on the presence or absence of vascular involvement. Demographic data, disease duration, disease activity, and medical treatments were recorded. Disease activity was evaluated using the Behçet's Disease Current Activity Form (BDCAF) and the Behçet's Syndrome Activity Scale (BSAS). The expression levels of miRNAs were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Results: The comparison of the clinical features of BD patients with and without vascular involvement revealed no significant difference. However, the expression levels of miR-195, miR-424, miR-10b, miR-103a-3p, and miR-542-3p were significantly higher in BD patients than in healthy controls (p<0.001, p<0.001, p=0.010, p<0.01, p=0.039, respectively). Moreover, the expression level of miR-195 was significantly higher in vasculo-Behçet patients than in the other groups (p=0.0318). However, no significant association was found between the expression levels of miR-195 and clinical activity. Conclusion: Our study results indicated elevated serum levels of miR-195 in BD patients, which may be associated with vascular involvement. Therefore, miR-195 could potentially serve as a biomarker for the diagnosis and monitoring of vasculo-Behçet's disease.

CYP2D6 copy number determination using digital PCR.

Background: CYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the "One-pot" single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5'UTR, intron 6, and exon 9 regions. Methods: Genomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0-6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5'UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA. Results: Overall, results between the two platforms and digestions methods were consistent. The "One-pot" digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference. Conclusion: The Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.

qPCR detection of Mycobacterium leprae DNA in urine samples of leprosy patients using the Rlep gene target.

Background: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, continues to pose a public health challenge in many parts of the world. Early and accurate diagnosis is crucial for effective treatment and prevention of disabilities associated with the disease. Molecular techniques such as PCR have demonstrated great potential as a diagnostic tool for directly detecting M. leprae DNA in different clinical samples, providing better sensitivity and specificity than conventional diagnostic techniques. The objective of this study was to measure the amount of M. leprae DNA in leprosy patients' urine samples using the Rlep gene target through qPCR. Methods: Different clinical samples such as smear, blood, and urine samples were collected from leprosy patients and healthy individuals. Leprosy patients were classified by the Ridley-Jopling classification. The Ziehl-Neelsen staining method was used for the slit skin smear (SSS) samples, and the bacteriological index (BI) was calculated for leprosy patients. DNA extraction and qPCR were performed for all three types of clinical samples using the Rlep gene target. Results: The Mycobacterial leprae DNA was successfully detected and quantified in all clinical samples across all types of leprosy among all the study groups using the Rlep gene (129 bp) target. The Rlep gene target was able to detect the presence of M. leprae DNA in 100% of urine, 96.1% of blood, and 92.2% of SSS samples of leprosy patients. Urine samples showed significant differences (p < 0.001) between the control and the different clinical forms and between borderline tuberculoid (BT) and pure neuritic leprosy (PNL) cases. There are significant differences in cycle threshold (Ct) values between control cases and clinical categories (p < 0.001), as well as specific differences within clinical categories (p < 0.001), reflecting the variability in bacterial load and detection sensitivity across different sample types and clinical manifestations of leprosy. Conclusion: Overall, this study's findings suggest that the qPCR technique can be used to detect M. leprae DNA in urine samples of leprosy patients using the Rlep gene target. It can also be used for diagnosing the disease and monitoring the effectiveness of anti-leprosy drugs, including multi-drug therapy (MDT), across various leprosy disease groups.

T-cell Large Granular Lymphocytic Leukemia with a STAT3 mutation successfully treated with Cord Blood Transplantation.

A 64-year-old woman presented with agranulocytosis, anemia, and bacteremia, leading to a diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL). A molecular analysis identified a signal transducer and activator of transcription 3 (STAT3) Y640F variant. Initial treatment with cyclophosphamide and prednisolone did not improve her condition, but serious infections were observed. The patient underwent cord blood transplantation (CBT) after preconditioning with fludarabine, busulfan, and total body irradiation, yielding a STAT3 Y640F variant disappearance, based on allele-specific quantitative polymerase chain reaction (AS-qPCR). In this case, CBT is a promising refractory T-LGLL treatment option, and the STAT3 Y640F variant AS-qPCR is a T-LGLL activity marker.

T-cell large granular lymphocytic leukemia with a STAT3 mutation successfully treated with cord blood transplantation.

A 64-year-old woman presented with agranulocytosis, anemia, and bacteremia, leading to a diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL). A molecular analysis identified a signal transducer and activator of transcription 3 (STAT3) Y640F variant. Initial treatment with cyclophosphamide and prednisolone did not improve her condition, but serious infections were observed. The patient underwent cord blood transplantation (CBT) after preconditioning with fludarabine, busulfan, and total body irradiation, yielding a STAT3 Y640F variant disappearance, based on allele-specific quantitative polymerase chain reaction (AS-qPCR). In this case, CBT is a promising refractory T-LGLL treatment option, and the STAT3 Y640F variant AS-qPCR is a T-LGLL activity marker.

Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer Chilo sacchariphagus (Lepidoptera: Pyralidae).

Chilo sacchariphagus (Lepidoptera: Pyralidae) is an economically important sugarcane pest. Although numerous studies were conducted on the physiological responses in C. sacchariphagus, little is known regarding the genes regulating these physiological processes. Gene expression analysis by qRT-PCR can offer a significant indication for functional gene studies. To our knowledge, the reference genes of C. sacchariphagus have not been screened or evaluated, which hinders the functional gene study. In the present study, the stability of seven reference genes (ß-ACT, GAPDH, BTF3, 28S, RPL7, EF1α, and SDHA) was evaluated in C. sacchariphagus under different experimental conditions, including tissues (antenna, head, thorax, abdomen, leg, and wing), temperatures (4 °C, 25 °C, and 37 °C) and sexes (male and female), through RefFinder, which integrates four algorithms (Normfinder, BestKeeper, ΔCt method, and geNorm). The findings suggested that the combination of ß-ACT and RPL7 is ideal to analyze gene expressions in different tissues and at distinct temperatures, and EF1α and SDHA were suitable reference genes for comparing gene expressions between sexes. Finally, the expression profiles of CsacPBP1 gene were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. This study represents the first kind in screening out suitable reference genes for gene expression analysis in C. sacchariphagus. Information from this study is poised to galvanize future inquiry into the gene expression of C. sacchariphagus, an economically important pest of sugarcane.

Study on the relationship between Methylation of Neuregulin (NRG) Gene and Cervical Carcinoma.

Objective: To investigate the relationship between the DNA methylation state of NRG1 promoter and its expression changes, and to analyze the clinical significance of its regulatory mechanism of DNA methylation in cervical carcinoma. Methods: This was a retrospective study. One-hundred and twenty patients from the Department of Gynecology of Cangzhou People's Hospital from September 2017 to September 2019 were selected, including 40 cases of cervical SCC, 40 cases of high grade squamous intraepithelial lesions(HSIL) and 40 cases of control cervical tissues. RT-qPCR, immunohistochemistry and DNA methylation-specific PCR(MSP) were used to detect the mRNA and protein expression of NRG1 and DNA methylation status in different tissue types. Results: Immunohistochemical results showed that the positive protein expression rate of NRG1 gene in the SCC group was lower than that in both HSIL and Control groups. RT-qPCR results showed that the mRNA gene of NRG1 gradually decreased in expression with the increase of cervical tissue lesions, with a statistically significant difference. Similarly, it also found that the mRNA expression level of NRG1 in the SCC group was independent of patients' age (p>0.05), but significantly correlated with tumor pathological staging, surgical pathology staging and lymphatic metastasis (p<0.05). Furthermore, methylation-specific PCR results revealed a significantly higher DNA methylation rate of NRG1 gene in the SCC group than in both HSIL and Control groups, with a statistically significant difference. Moreover, the methylation degree of NRG1 gene in SCC tissues was negatively correlated with its mRNA expression (p<0.05). Conclusions: Abnormal DNA hypermethylation of NRG1 gene inhibits the expression of mRNA and protein in the progression of cervical tissue from normal to cancerous state, which is involved in the occurrence and development of cervical carcinoma.

Quantitative PCR-based high-sensitivity detection of HBV-DNA levels reflects liver function deterioration in patients with hepatitis B virus-related cirrhosis.

OBJECTIVES: To investigate the clinical implication of quantitative polymerase chain reaction (PCR)-based high-sensitivity detection of hepatitis B virus (HBV)-DNA levels in patients with HBV-related liver cirrhosis (LC). METHODS: From January 2020 to December 2022, 100 fasting serum samples were collected and retrospectively analyzed from patients with treated HBV-related LC attending the Suzhou Hospital of Integrated Traditional Chinese and Western Medicine and Suzhou Guangci Cancer Hospital. Patients were divided into a negative group (HBV-DNA < 20 IU/mL) and a positive group (HBV-DNA ≥ 20 IU/mL) according to their high-sensitivity HBV-DNA test results. The clinical characteristics and serological indicators of the two groups were compared, mainly including gender, age, liver function [total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL)], lipids [total cholesterol (TC) and triglycerides (TG)], platelets (PLT), five serum liver fibrosis markers [cholyglycine (CG), hyaluronic acid (HA), laminin (LN), precollagen type III (PCIII), and type IV collagen (IV-C)], serum gastrointestinal tumor markers [α-fetoprotein (AFP) and carcinoembryonic antigen (CEA)], and hepatitis B surface antigen (HBsAg). The differences between the two groups in terms of liver function Child-Pugh grades and the incidence of hepatocellular carcinoma (HCC) were also compared. RESULTS: There were 39 patients in the positive group, including 29 males and 10 females, and 61 patients in the negative group, including 38 males and 23 females, with no statistically significant differences in gender and age distribution between the two groups (P > 0.05). The levels of serological indicators (TP, ALB, AST, GGT, ALP, TBIL, DBIL, IBIL, TC, TG, PLT, CG, HA, LN, PCIII, IV-C, AFP, CEA, and HBsAg) in both groups showed no significant differences (P > 0.05), but the ALT level in the positive group was higher than that in the negative group (P < 0.0001). The positive group had worse Child-Pugh grades and higher HCC incidence compared to the negative group (P < 0.0001, P = 0.028). CONCLUSIONS: Patients with HBV-related LC and HBV-DNA ≥ 20 IU/mL have higher serum ALT levels, worse liver function Child-Pugh grades, and higher HCC incidence than those with HBV-DNA < 20 IU/mL. High-sensitivity HBV-DNA quantification can reflect the deterioration of liver function in patients with HBV-related LC to some extent.

Gene Expression and Phenotypic Assessment of Egg Quality across Developmental Stages of Atlantic Cod throughout the Spawning Season.

Egg quality in fishes is commonly determined by fertilisation success and cleavage patterns as a phenotypic outcome of underlying regulatory mechanisms. Although these phenotypic estimators of egg quality are useful in farming conditions, these "good quality" egg batches do not always translate to good larval growth and survival. The identification of genes involved in embryonic development may help find links between genetic factors of maternal origin and egg quality. Herein, the relative expression of seven stage-specific developmental genes of Atlantic cod was analysed using quantitative PCR to understand the function during embryogenesis and its relationship with egg quality. Genes ccnb2 and pvalb1 showed significant differential expression between developmental stages and significant upregulation from blastula and somite stages, respectively. The comparison of spawning batches showed that the relative gene expression of genes ccnb2, acta, tnnt3 and pvalb1 was significantly higher from the middle of the spawning season where phenotypic quality estimators establish the best egg quality. Moreover, a positive significant correlation was observed between quality estimators based on egg morphology and the genetic expression of genes acta and acta1 during somitogenesis. This study suggests that the combination of quality estimators, genetics and batch timing could help optimise reproductive protocols for commercial stocks of Atlantic cod.

Target screening using RNA interference in the sheep abomasal nematode parasite Haemonchus contortus.

RNA interference (RNAi) on parasitic nematodes has been described as a valuable tool for screening putative targets that could be used as novel drug and/or vaccine candidates. This study aimed to set up a pipeline to identify potential targets using RNAi for vaccine/anti-parasite therapy development against Haemonchus contortus, a blood-feeding abomasal nematode parasite. The available H. contortus sequence data was mined for targets, which were tested for essentiality using RNAi electroporation assays. A total of 56 genes were identified and tested for knockdown using electroporation of first-stage larvae (L1) H. contortus with the target double-stranded RNA. Electroporation of L1 proved to be effective overall; 17 targets had a strong phenotype and significant reduction in alive H. contortus, and another 24 had a moderate phenotype with a significant reduction in larvae development. A total of 28 targets showed a significant reduction in the development of H. contortus larvae to the infective stage (L3) following the RNAi assay. Down-regulation of target transcript levels was evaluated in some targets by semi-quantitative PCR. Four out of five genes tested showed complete knockdown of mRNA levels via semi-quantitative PCR, whereas the knockdown was partial for one. In conclusion, the results indicate that the RNAi pathway is confirmed in H. contortus and that several target genes have the potential to be investigated further as possible vaccine candidates.

Prognostic value of oxidative phosphorylation-related genes in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is the most prevalent malignancies worldwide. Recently, oxidative phosphorylation (OXPHOS) has received extensive concern as an emerging target in antitumor therapy. However, the OXPHOS-involved underlying genes and clinical utilization in HCC remain worth exploring. The present research aimed to create an OXPHOS-relevant signature in HCC. PATIENTS AND METHODS: In this study, the prognostic signature genes linked with OXPHOS were identified, and prognostic models were built using least absolute shrinkage and selection operator (LASSO) cox regression analysis. Furthermore, the combination study of immune microenvironment and signature genes looked into the involvement of immune cells in signature-based genes in HCC. Following that, chemotherapeutic drug sensitivity and immunotherapy analysis was implemented to predict clinical efficacy in HCC patients. Finally, clinical samples were collected to measure the expression of OXPHOS-related signature genes. RESULTS: Following a series of screens, six prognostic signature genes related with OXPHOS were identified: MRPS23, MPV17, MAPK3, IGF2BP2, CDK5, and IDH2, on which a risk model was built. The findings revealed a significant drop in the survival rate of HCC patients as their risk score increased. Meanwhile, independent prognostic study demonstrated that the risk score could accurately identify HCC patients. Immuno-microenvironmental correlation research suggested that the prognostic characteristics could serve as a reference index for both immunotherapy and chemotherapy. Finally, RT-qPCR exhibited a trend in signature gene expression that was consistent with the results. CONCLUSION: In this study, a total of six prognostic genes associated with OXPHOS were selected and a prognostic model was constructed, providing an essential reference for the study of OXPHOS in HCC.

Multiple antibiotic resistance and herbicide catabolic profiles of bacteria isolated from Lake Villarrica surface sediments (Chile).

Antibiotics and herbicides are contaminants of emerging concern in aquatic environments. Lake Villarrica is a relevant freshwater body in Chile and was recently designated a 'saturated nutrient zone'. Here, we investigated the occurrence of multiple antibiotic resistance (MAR) and herbicide catabolic profiles among bacteria present in the surface sediments of Lake Villarrica. The occurrence of antibiotic-resistant genes (ARGs; blaTEM, catA and tetM) and herbicide-catabolic genes (HCGs; phnJ and atzA) was investigated by qPCR. Subsequently, the presence of culturable bacteria with multiple resistance to amoxicillin (AMX), chloramphenicol (CHL) and oxytetracycline (OXT) was studied. Forty-six culturable MAR (AMX + CHL + OXT) strains were isolated and characterized with respect to their resistance to 11 antibiotics by using a disc diffusion assay and testing their ability to use herbicides as a nutrient source. qPCR analyses revealed that ARGs and HCGs were present in all sediment samples (101 to 103 gene copies g-1), with significant (P ≤ 0.05) higher values in sites near Villarrica city and cattle pastures. The plate method was used to recover MAR isolates from sediment (103-106 CFU g-1), and most of the 46 isolates also showed resistance to oxacillin (100%), cefotaxime (83%), erythromycin (96%) and vancomycin (93%). Additionally, 54 and 57% of the MAR isolates were able to grow on agar supplemented (50 mg L-1) with atrazine and glyphosate as nutrient sources, respectively. Most of the MAR isolates were taxonomically close to Pseudomonas (76.1%) and Pantoea (17.4%), particularly those isolated from urbanized sites (Pucón city). This study shows the presence of MAR bacteria with herbicide catabolic activity in sediments, which is valuable for conservation strategies and risk assessments of Lake Villarrica. However, major integrative studies on sediments as reservoirs or on the fate of MAR strains and traces of antibiotics and herbicides as a result of anthropic pressure are still needed.

Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community.

Amidst the COVID-19 pandemic, the Polytechnic University of Setúbal (IPS) used its expertise in molecular genetics to establish a COVID-19 laboratory, addressing the demand for community-wide testing. Following standard protocols, the IPS COVID Lab received national accreditation in October 2020 and was registered in February 2021. With the emergence of new SARS-CoV-2 variants and safety concerns for students and staff, the lab was further challenged to develop rapid and sensitive diagnostic technologies. Methodologies such as sample-pooling extraction and multiplex protocols were developed to enhance testing efficiency without compromising accuracy. Through Real-Time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) analysis, the effectiveness of sample pooling was validated, proving to be a clear success in COVID-19 screening. Regarding multiplex analysis, the IPS COVID Lab developed an in-house protocol, achieving a sensitivity comparable to that of standard methods while reducing operational time and reagent consumption. This approach, requiring only two wells of a PCR plate (instead of three for samples), presents a more efficient alternative for future testing scenarios, increasing its throughput and testing capacity while upholding accuracy standards. The lessons learned during the SARS-CoV-2 pandemic provide added value for future pandemic situations.

Evaluation of Nanonanoliposomal Curcumin on Cutaneous Leishmaniasis Skin Lesions Caused by Leishmania major in BALB/c Mice.

Background: Curcumin is an extract of rhizome turmeric (diferuloylmethane), with antioxidant, anti-inflammatory, antimicrobial, and anti-parasitic properties, which making it a potential candidate for the treatment of leishmaniasis. The aim of the presented study was to evaluate curcumin as possible candidate for treatment of cutaneous leishmaniasis. Methods: We investigated the physicochemical properties and anti-leishmanial effects of nanoliposomal curcumin (40, 80, and 120 µM) in Leishmania major (MRHO/IR/75/ER) infected BALB/c mice at the faculty of Veterinary Medicinem University of Tehran, Iran. For this aim, L. major promastigotes (MHROM/IR/75/ER) at stationary phase (2×106) were inoculated sub-cutaneously into the upper area of the tail in BALB/c mice (six groups, n= 10 per group). For evaluation of nanoliposomal curcumin, the zeta potential, particle size and stability of nanoliposomal curcumin was determined. Furthermore, the anti-leishmanial effects of nanoliposomal curcumin formulation on the lesion sizes was determined and the parasite burden in the leishmania induced lesion was performed using semi quantitative PCR. Results: Treatment of L. major infected BALB/c mice with nanoliposomal curcumin led to a reduction in the kinetic of the skin lesion size development. The semi quantitative PCR analysis of DNA extracted from the lesions showed reduction of parasite burden. The most effective treatment could be found in 80 µM nanoliposomal curcumin. Treatment with Glucantime, as a positive control, also showed a nearly similar effect compared to the effect of 80 µM nanoliposomal curcumin. Conclusion: Nanoliposomal curcumin could be considered as a potential drug against cutaneous leishmaniasis caused by L. major in susceptible animal models.