qPCR Assay Research Articles

Cell-Free Nuclear and Mitochondrial DNA as Potential Biomarkers for Assessing Sepsis Severity.

Sepsis continues to be a significant public health challenge despite advances in understanding its pathophysiology and management strategies. Therefore, this study evaluated the value of cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) for assessing the severity and prognosis of sepsis. Ninety-four patients were divided into three groups: infection (n = 32), sepsis (n = 30), and septic shock (n = 32). Plasma samples were collected at the time of diagnosis, and cfDNA concentrations were determined by qPCR assay. The results showed that plasma cfDNA levels increased with the severity of the disease. To distinguish between patients with infection and those with sepsis, the biomarker L1PA290 achieved the highest AUC of 0.817 (95% CI: 0.725-0.909), demonstrating a sensitivity of 77.0% and a specificity of 79.3%. When cf-nDNA was combined with the SOFA score, there was a significant improvement in the AUC (0.916 (0.853-0.979)), sensitivity (88.1%), and specificity (80.0%). Moreover, patients admitted to the ICU after being diagnosed with sepsis had significantly higher cf-nDNA concentrations. In patients admitted to the ICU, combining cf-nDNA with the SOFA score yielded an AUC of 0.753 (0.622-0.857), with a sensitivity of 95.2% and a specificity of 50.0%. cfDNA can differentiate between patients with infection and those with sepsis. It can also identify patients who are likely to be admitted to the ICU by predicting those with indications for intensive care, suggesting its potential as a biomarker for sepsis.

The thrombin receptor PAR4 supports visceral adipose tissue inflammation.

Thrombin inhibition suppresses adiposity, WAT inflammation and metabolic dysfunction in mice. Protease-activated receptor (PAR)1 does not account for thrombin-driven obesity, so we explored the culprit role of PAR4 in this context. Male WT and PAR-4-/- mice received a high fat diet (HFD) for 8 weeks, WT controls received standard chow. Body fat was quantified by NMR. Epididymal WAT was assessed by histology, immunohistochemistry, qPCR and lipase activity assay. 3T3-L1 preadipocytes were differentiated ± thrombin, acutely stimulated ± PAR4 activating peptide (AP) and assessed by immunoblot, qPCR and U937 monocyte adhesion. Epicardial adipose tissue (EAT) from obese and lean patients was assessed by immunoblot. PAR4 was upregulated in mouse WAT under HFD. PAR4-/- mice developed less visceral adiposity and glucose intolerance under HFD, featuring smaller adipocytes, fewer macrophages and lower expression of adipogenic (leptin, PPARγ) and pro-inflammatory genes (CCL2, IL-1β) in WAT. HFD-modified activity and expression of lipases or perilipin were unaffected by PAR4 deletion. 3T3-L1 adipocytes differentiated with thrombin retained Ki67 expression, further upregulated IL-1β and CCL2 and were more adhesive for monocytes. In mature adipocytes, PAR4-AP increased phosphorylated ERK1/2 and AKT, upregulated Ki67, CCl2, IL-β and hyaluronan synthase 1 but not TNF-α mRNA, and augmented hyaluronidase-sensitive monocyte adhesion. Obese human EAT expressed more PAR4, CD68 and CD54 than lean EAT. PAR4 upregulated in obesity supports adipocyte hypertrophy, WAT expansion and thrombo-inflammation. The emerging PAR4 antagonists provide a therapeutic perspective in this context beyond their canonical antiplatelet action.

Overexpression of ZNF169 promotes the growth and proliferation of colorectal cancer cells via the upregulation of ANKZF1.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The 5‑year survival rate of patients diagnosed with the early stages of the disease is markedly higher than that of patients in the advanced stages. Therefore, identifying novel biomarkers and drug targets for CRC is critical for clinical practice. Zinc finger protein 169 (ZNF169) is a crucial transcription factor, and its role in CRC remains to be explored. The present study aimed to investigate the clinical relevance, function and underlying mechanisms of ZNF169 in CRC growth and proliferation. The Cancer Genome Atlas (TCGA) database was utilized to analyze the clinical relevance of ZNF169 in patients with CRC. Immunohistochemical staining was performed on tissue samples from patients with CRC to detect the expression of ZNF169. The HCT‑116, HT‑29 and RKO cell lines were employed for in vitro experiments. The overexpression and knockdown of ZNF169 were achieved by transfecting the cells with lentivirus and small interfering RNAs, respectively. Cell Counting Kit‑8, colony formation and EdU staining assays were applied to investigate the function of ZNF169 in CRC cells. Dual luciferase activity and chromatin immunoprecipitation (ChIP)‑quantitative PCR (qPCR) assays were performed to identify the regulatory effects of ZNF169 on the ankyrin repeat and zinc‑finger domain‑containing 1 (ANKZF1; also known as ZNF744) gene. Reverse transcription‑quantitative PCR and western blot analysis were performed to measure mRNA and protein expression, respectively. The analysis of TCGA data revealed a positive correlation between ZNF169 and ANKZF1, with the overexpression of ANKZF1 being associated with a poor prognosis of patients with CRC. The experimental results demonstrated that ZNF169 was expression upregulated in CRC tissue compared with that in normal colon tissue. Gain‑of‑function and loss‑of‑function experiments revealed that ZNF169 enhanced the intensity of EdU staining, promoting the growth and proliferation of CRC cells. Furthermore, the overexpression of ZNF169 potentiated the transcriptional activity of the ANKZF1 gene, while the knockdown of ZNF169 produced the opposite results. ChIP‑qPCR confirmed the interaction between ZNF169 and the promoter sequence of ANKZF1. Rescue experiments revealed that ZNF169 accelerated CRC cell growth and proliferation through the upregulation of ANKZF1. Furthermore, there was a positive correlation identified between ZNF169 and ANKZF1, and upregulation of ANKZF1 expression was associated with the poor prognosis of patients with CRC. On the whole, the present study demonstrates that ZNF169 contributes to CRC malignancy by potentiating the expression of ANKZF1. Thus, the regulation of ZNF169 and/or ANKZF1 expression may represent a viable strategy for the treatment patients with CRC with a high expression of ZNF169.

Molecular investigation and genetic characterization of feline leukemia virus (FeLV) in cats referred to a veterinary teaching hospital in Northern Italy

Feline leukemia virus (FeLV) is responsible for feline leukemia syndrome in domestic cats. The prevention and control of disease caused by FeLV are primarily based on vaccination and identification and isolation of infected subjects. Antigen diagnostic methods, which are the most widely used in clinical practices, can be associated to molecular tests to characterize the FeLV detected. In this study, a quantitative SYBR Green Real-Time PCR (qPCR) assay was used to detect FeLV proviral DNA in blood samples from antigen positive cats referred to a veterinary teaching hospital in Northern Italy in 2018–2021. To genetically characterize the identified viruses, a portion of the viral envelope (env) gene was amplified using six different end-point PCRs and sequenced. Twenty-two of 26 (84.6%) cats included in the study tested positive by qPCR assay. This suggests a high performance of the qPCR adopted but further studies are required to investigate the cause of discordant results between the antigen test and qPCR in four cats. From env gene analysis, 15/22 qPCR-positive cats were infected by FeLV subtype A and 5/15 shown coinfection with subtype B.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

ObjectiveRecombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. MethodsRecombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. ResultsThe M-RAP method had sensitivity rates of 1, 10, and 1 copies/μL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively (P < 0.05). ConclusionAn M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Enhancing pressure ulcer healing and tissue regeneration by using N-acetyl-cysteine loaded carboxymethyl cellulose/gelatin/sodium alginate hydrogel.

Prolonged pressure on the skin can result in pressure ulcers, which may lead to serious complications, such as infection and tissue damage. In this study, we evaluated the effect of a carboxymethyl cellulose/gelatin/sodium alginate (CMC/Gel/Alg) hydrogel containing N-acetyl-cysteine (NAC) on the healing of pressure ulcers. Pressure ulcers were induced by applying a magnet to the dorsum of rat skin. The wounds were then treated with sterile gauze, ChitoHeal Gel®, and CMC/Gel/Alg hydrogel dressings with or without NAC for the other groups. We evaluated the morphology, weight loss, swelling, rheology, blood compatibility, cytocompatibility, antioxidant capacity, and wound scratch of the prepared hydrogel. MTT assay revealed that the optimum concentration of NAC was 5mg/ml, which induced higher cell proliferation and viability. Results of the histopathological evaluation showed increased wound closure, and complete re-epithelialization in the hydrogel-containing NAC group compared to the other groups. The CMC/Gel/Alg/5mg/ml NAC hydrogel dressing showed 84% wound closure at 14days after treatment. Immunohistochemical results showed a decrease in the level of TNF-α on day 14 compared day 7. Results of the qPCR assay revealed that NAC hydrogel increased the expression of Collagen type I and TGF-β1 and decreased MMP2 and MMP9 mRNA on the 14th day. The results suggest that the CMC/Gel/Alg/5mg/ml NAC hydrogel with antioxidant properties is an appropriate dressing for wound healing.

Revealing the mechanism of ethyl acetate extracts of Semen Impatientis against prostate cancer based on network pharmacology and transcriptomics

Ethnopharmacological relevanceProstate cancer (PCa) is the most common malignancy of the male genitourinary system and currently lacks effective treatment. Semen Impatientis, the dried ripe seed of Impatiens balsamina L., is described by the Chinese Pharmacopoeia as a traditional Chinese medicine (TCM) and is used in clinical practice to treat tumors, abdominal masses, etc. In our previous study, the ethyl acetate extracts of Semen Impatientis (EAESI) was demonstrated to be the most effective extract against PCa among various extracts. However, the biological effects of EAESI against PCa in vivo and the specific antitumor mechanisms involved remain unknown. Aim of the studyIn this study, we aimed to investigate the antitumor effect of EAESI on PCa in vitro and in vivo by performing network pharmacology analysis, transcriptomic analysis, and experiments to explore and verify the underlying mechanisms involved. Materials and methodsThe antitumor effect of EAESI on PCa in vitro and in vivo was investigated via CCK-8, EdU, flow cytometry, and wound healing assays and xenograft tumor models. Network pharmacology analysis and transcriptomic analysis were employed to explore the underlying mechanism of EAESI against PCa. Activating transcription factor 3 (ATF3) and androgen receptor (AR) were confirmed to be the targets of EAESI against PCa by RT‒qPCR, western blotting, and rescue assays. In addition, the interaction between ATF3 and AR was assessed by coimmunoprecipitation, immunofluorescence, and nuclear–cytoplasmic separation assays. ResultsEAESI decreased cell viability, inhibited cell proliferation and migration, and induced apoptosis in AR+ and AR− PCa cells. Moreover, EAESI suppressed the growth of xenograft tumors in vivo. Network pharmacology analysis revealed that the hub targets of EAESI against PCa included AR, AKT1, TP53, and CCND1. Transcriptomic analysis indicated that activating transcription factor 3 (ATF3) was the most likely critical target of EAESI. EAESI downregulated AR expression and decreased the transcriptional activity of AR through ATF3 in AR+ PCa cells; and EAESI promoted the expression of ATF3 and exerted its antitumor effect via ATF3 in AR+ and AR− PCa cells. ConclusionsEAESI exerts good antitumor effects on PCa both in vitro and in vivo, and ATF3 and AR are the critical targets through which EAESI exerts antitumor effects on AR+ and AR− PCa cells.

Fisetin Suppresses Atherosclerosis by Inhibiting Ferroptosis-Related Oxidative Stress in Apolipoprotein E Knockout Mice

Introduction: Fisetin has been demonstrated to inhibit the occurrence of atherosclerosis; however, the mechanism of fisetin suppressing atherosclerosis remains elusive. Methods: The function of fisetin in the inhibition of atherosclerosis was evaluated by hematoxylin and eosin and Oil Red O staining in ApoE−/− mice. Molecular biomarkers of atherosclerosis progression were detected by Western blot and qPCR. Moreover, the inhibition of atherosclerosis on oxidative stress and ferroptosis was evaluated by immunofluorescence staining, qPCR, and Western blot assays. Results: The obtained results showed that serum lipid was attenuated and consequentially the formation of atherosclerosis was also suppressed by fisetin in ApoE−/− mice. Exploration of the mechanism revealed that molecular biomarkers of atherosclerosis were decreased under fisetin treatment. The level of reactive oxygen species and malondialdehyde declined, while the activity of superoxide dismutases and glutathione peroxidase was increased under the fisetin treatment. Additionally, the suppressor of ferroptosis, glutathione peroxidase 4 proteins, was elevated. The ferritin was decreased in the aortic tissues treated with fisetin. Conclusions: In summary, fisetin attenuated the formation of atherosclerosis through the inhibition of oxidative stress and ferroptosis in the aortic tissues of ApoE−/− mice.

Authenticity Determination of Goat Cheese In Mexico Using qPCR Assay

Authenticity Determination of Goat Cheese In Mexico Using qPCR Assay

Comparative evaluation of three real-time polymerase chain reaction assays to detect Myxobolus cerebralis.

We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc. Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays. The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book. The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc.

N6-Methyladenosine–Modified KREMEN2 Promotes Tumorigenesis and Malignant Progression of High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) remains the most lethal female cancer by far. Herein, clinical HGSOC samples had higher N6-methyladenosine (m6A) modification than normal ovarian tissue, and its dysregulation had been reported to drive aberrant transcription and translation programs. However, Kringle-containing transmembrane protein 2 (KREMEN2) and its m6A modification have not been fully elucidated in HGSOC. In this study, the data from the high-throughput messenger RNA (mRNA) sequencing of clinical samples were processed using the weighted correlation network analysis and functional enrichment analysis. Results revealed that KREMEN2 was a driver gene in the tumorigenesis of HGSOC and a potential target of m6A demethylase fat-mass and obesity-associated protein (FTO). KREMEN2 and FTO levels were upregulated and downregulated, respectively, and correlation analysis showed a significant negative correlation in HGSOC samples. Importantly, upregulated KREMEN2 was remarkably associated with lymph node metastasis, distant metastasis, peritoneal metastasis, and high International Federation of Gynecology and Obstetrics stage (Ⅲ/Ⅳ), independent of the age of patients. KREMEN2 promoted the growth of HGSOC in vitro and in vivo, which was dependent on FTO. The methylated RNA immunoprecipitation qPCR and RNA immunoprecipitation assays were performed to verify the m6A level and sites of KREMEN2. FTO overexpression significantly decreased m6A modification in the 3′ and 5′ untranslated regions of KREMEN2 mRNA and downregulated its expression. In addition, we found that FTO-mediated m6A modification of KREMEN2 mRNA was recognized and stabilized by the m6A reader IGF2BP1 rather than by IGF2BP2 or IGF2BP3. This study highlights the m6A modification of KREMEN2 and extends the importance of RNA epigenetics in HGSOC.

An On-Farm Workflow for Predictive Management of Paralytic Shellfish Toxin-Producing Harmful Algal Blooms for the Aquaculture Industry.

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire's buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7-106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L-1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture.

Rapid detection and molecular epidemiology of β-lactamase producing Enterobacteriaceae isolated from food animals and in-contact humans in Nigeria.

The emergence and spread of β-lactamase-producing Enterobacteriaceae poses a significant threat to public health, necessitating the rapid detection and investigation of the molecular epidemiology of these pathogens. We modified a multiplex real-time (RT)-PCR to concurrently detect β-lactamase genes (blaCTX-M, blaTEM, and blaSHV) and Enterobacteriaceae 16S ribosomal RNA. qPCR probes and primers were validated using control isolates, and the sensitivity and specificity assessed. The optimised multiplex qPCR was used to screen 220 non-clinical Enterobacteriaceae from food animals and in-contact humans in Southeast Nigeria selected on cefotaxime-supplemented agar plates. Binary logistic regression was used to explore factors associated with the presence of the blaTEM and blaSHV genes in these isolates, and a subset of isolates from matched sampling sites and host species were whole genome sequenced, and their antimicrobial resistance (AMR) and plasmid profiles determined. The sensitivity and specificity of the qPCR assay was 100%. All isolates (220/220) were positive for Enterobacteriaceae ribosomal 16S rRNA and blaCTX-M, while 66.4% (146/220) and 9% (20/220) were positive for blaTEM and blaSHV, respectively. The prevalence of blaTEM and blaSHV varied across different sampling sites (farm, animal market and abattoirs). Isolates from Abia state were more likely to harbour blaTEM (OR = 2.3, p = 0.04) and blaSHV (OR = 5.12,p = 0.01) than isolates from Ebonyi state; blaTEM was more likely to be detected in isolates from food animals than humans (OR = 2.34, p = 0.03), whereas the reverse was seen for blaSHV (OR = 7.23, p = 0.02). Furthermore, Klebsiella and Enterobacter isolates harboured more AMR genes than Escherichia coli, even though they were isolated from the same sample. We also identified pan resistant Klebsiella harbouring resistance to ten classes of antimicrobials and disinfectant. Therefore, we recommend ESKAPE pathogens are included in AMR surveillance in future and suggest qPCRs be utilised for rapid screening of Enterobacteriaceae from human and animal sources.

Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices

BackgroundMelioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence.MethodsAn extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls.ResultsA novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil.ConclusionsThe findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

Anopheles sacharovi in Italy: first record of the historical malaria vector after over 50 years

BackgroundAnopheles sacharovi, a member of the Anopheles maculipennis complex, was a historical malaria vector in Italy, no longer found since the last report at the end of 1960s. In September 2022, within the Surveillance Project for the residual anophelism, a single specimen of An. maculipennis sensu lato collected in Lecce municipality (Apulia region) was molecularly identified as An. sacharovi. This record led to implement a targeted entomological survey in September 2023. MethodsInvestigation was conducted in the areas around the first discovery, focusing on animal farms, riding stables and potential breeding sites. Adult and immature mosquitoes were collected, using active search or traps, in several natural and rural sites. Mosquitoes belonging to An. maculipennis complex were identified morphologically and molecularly by a home-made routine quantitative polymerase chain reaction (qPCR) assay, developed specifically for the rapid identification of An. labranchiae, and, when necessary, by amplification and sequencing of the ITS-2 molecular marker.ResultsOut of the 11 sites investigated, 6 were positive for Anopheles presence. All 20 An. maculipennis s.l. (7 adults, 10 larvae and 3 pupae) collected in the areas were identified as An. sacharovi by ITS-2 sequencing.ConclusionsThe discovery of An. sacharovi, considered to have disappeared from Italy for over 50 years, has a strong health relevance and impact, highlighting an increase in the receptivity of the southern areas. As imported malaria cases in European countries are reported every year, the risk of Plasmodium introduction by gametocyte carriers among travellers from endemic countries should be taken into greater consideration. Our findings allow rethinking and building new models for the prediction and expansion of introduced malaria. Furthermore, to prevent the risk of reintroduction of the disease, the need to strengthen the surveillance of residual anophelism throughout the South should be considered.Graphical

Identification of a molecular network regulated by multiple ASD high risk genes.

Genetic sequencing has identified high-confidence ASD risk genes with loss-of-function mutations. How the haploinsufficiency of distinct ASD risk genes causes ASD remains to be elucidated. In this study, we examined the role of four top-ranking ASD risk genes, ADNP, KDM6B, CHD2, and MED13, in gene expression regulation. ChIP-seq analysis reveals that gene targets with the binding of these ASD risk genes at promoters are enriched in RNA processing and DNA repair. Many of these targets are found in ASD gene database (SFARI), and are involved in transcription regulation and chromatin remodeling. Common gene targets of these ASD risk genes include a network of high confidence ASD genes associated with gene expression regulation, such as CTNNB1 and SMARCA4. We further directly examined the transcriptional impact of the deficiency of these ASD risk genes. Our mRNA profiling with qPCR assays in cells with the knockdown of Adnp, Kdm6b, Chd2 or Med13 has revealed an intricate pattern of their cross-regulation, as well as their influence on the expression of other ASD genes. In addition, some synaptic genes, such as Snap25 and Nrxn1, are strongly regulated by deficiency of the four ASD risk genes, which could be through the direct binding at promoters or indirectly through the targets like Ctnnb1 or Smarca4. The identification of convergent and divergent gene targets that are regulated by multiple ASD risk genes will help to understand the molecular mechanisms underlying common and unique phenotypes associated with haploinsufficiency of ASD-associated genes.

A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Ablation of IFNγ in myeloid cells suppresses liver inflammation and fibrogenesis in mice with hepatic small heterodimer partner (SHP) deletion

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is a common complication of obesity and, in severe cases, progresses to metabolic dysfunction-associated steatohepatitis (MASH). Small heterodimer partner (SHP) is an orphan member of the nuclear receptor superfamily and regulates metabolism and inflammation in the liver via a variety of pathways. In this study, we investigate the molecular foundation of MASH progression in mice with hepatic SHP deletion and explore possible therapeutic means to reduce MASH. MethodsHepatic SHP knockout mice (SHPΔhep) and their wild-type littermates (SHPfl/fl) of both sexes were fed a fructose diet for 14 weeks and subjected to an oral glucose tolerance test. Then, plasma lipids were determined, and liver lipid metabolism and inflammation pathways were analyzed with immunoblotting, RNAseq, and qPCR assays. To explore possible therapeutic intersections of SHP and inflammatory pathways, SHPΔhep mice were reconstituted with bone marrow lacking interferon γ (IFNγ−/−) to suppress inflammation. ResultsHepatic deletion of SHP in mice fed a fructose diet decreased liver fat and increased proteins for fatty acid oxidation and liver lipid uptake, including UCP1, CPT1α, ACDAM, and SRBI. Despite lower liver fat, hepatic SHP deletion increased liver inflammatory F4/80+ cells and mRNA levels of inflammatory cytokines (IL-12, IL-6, Ccl2, and IFNγ) in both sexes and elevated endoplasmic reticulum stress markers of Cox2 and CHOP in female mice. Liver bulk RNAseq data showed upregulation of genes whose protein products regulate lipid transport, fatty acid oxidation, and inflammation in SHPΔhep mice. The increased inflammation and fibrosis in SHPΔhep mice were corrected with bone marrow-derived IFNγ−/− myeloid cell transplantation. ConclusionHepatic deletion of SHP improves fatty liver but worsens hepatic inflammation possibly by driving excess fatty acid oxidation, which is corrected by deletion of IFNγ specifically in myeloid cells. This suggests that hepatic SHP limits fatty acid oxidation during fructose diet feeding but, in doing so, prevents pro-MASH pathways. The IFNγ-mediated inflammation in myeloid cells appears to be a potential therapeutic target to suppress MASH.

Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

The effect of LINC9137 targeting miR-140-3p-NKAIN3 signal axis on the development of goose testis sertoli cells

Sertoli cells (SC) are a type of important cells in the testes, which can provide transport proteins, regulatory proteins, growth factors, and other cytokines for the spermatogenic process. They participate in the regulation of the maturation and differentiation of spermatogenic cells and play an important supporting role in the migration, proliferation, and differentiation of germ cells at all levels in the testes. Previous studies found differential expression of LINC9137, miR-140-3p, and Sodium/Potassium Transporting ATPase Interacting 3 (NKAIN3) genesin high and low sperm motility goose testicular tissues. This study investigated the effects of the LINC9137-miR-140-3p-NKAIN3 signal axis on the proliferation and apoptosis of goose testicular sertoli cells at the cellular level, respectively. The results showed that through acridine orange staining, oil red O staining, Alkaline phosphatase (AKP) staining, and RT qPCR assay, it was comprehensively identified that the cultured testicular sertoli cells were purified in vitro. Through the dual luciferase activity detection test, it was found that LINC9137 has a targeted binding site with miR-140-3p and NKAIN3. In addition, this study found that overexpression of miR-140-3p significantly inhibited the expression of LINC9137 and NKAIN3 in sertoli cells, and their expression was significantly increased when miR-140-3p was interfered with. By measuring cell proliferation activity and apoptosis related gene expression, it was found that overexpression of LINC9137 decreased cell proliferation activity (P > 0.05), while the expression level of apoptosis factor Bcl2 Associated X Protein (Bax)/B-cell lymphoma-2 (Bcl2) increased (P > 0.05). On the contrary, when interfering with LINC9137, the cell proliferation activity of sertoli cells was significantly increased (P < 0.01), and the expression level of apoptosis factor Bax/Bcl2 was significantly reduced (P < 0.05); The effect of miR-140-3p on the proliferation and apoptosis of sertoli cells is opposite to that of LINC9137. Meanwhile, this study co transfected overexpressed LINC9137 and miR-140-3p plasmids into sertoli cells, and found that the effect of LINC9137 overexpression on supporting cell proliferation was weakened by miR-140-3p. This study elucidates the role and function of the LINC9137 miR-140-3p-NKAIN3 signaling axis in the development of goose testes and spermatogenesis, establishes a regulatory network related to spermatogenesis, and provides a theoretical basis for studying the genetic regulation of goose spermatogenesis.