Fluorescent PCR Research Articles

A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening

BackgroundGenome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. ResultsHere, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1–5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene.ConclusionsWe have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.

Roles and mechanisms of m6A modification regulating RP11-426A6.5 in laryngeal squamous cell carcinoma

Objective: To investigate the roles of N6-methyladenosine (m6A) modification in regulating RP11-426A6.5 in the development of laryngeal squamous cell carcinoma (LSCC). Methods: The methylation and expression levels of lncRNAs were identified and important lncRNAs were screened utilizing long non-coding RNA (lncRNA) m6A methylation microarray. Cancer and para cancer tissue samples were taken from 48 LSCC patients hospitalized to the Department of Otolaryngology-Head and Neck Surgery of the Second Affiliated Hospital of Harbin Medical University between January and September 2017. Expression profiling microarray was performed in 3 of 48 LSCC samples, and methylated RNA immunoprecipitation-quantitative PCR (MeRIP-qPCR) and quantitative real-time fluorescent PCR (qRT-PCR) were performed in the remaining 45 LSCC samples to verify the m6A modification and expression levels of RP11-426A6.5. Correlations between RP11-426A6.5 and clinical factors were anlysed. Laryngeal cancer cell line with low expression of RP11-426A6.5 was created in vitro using RNA interference (RNAi) technology. The 5-Ethynyl-2'-deoxyuridine (EdU) cell proliferation experiment, wound healing experiment, and transwell invasion experiment were used respectively to measure the proliferation, migration, and invasion of LSCC cells. The effect of RP11-426A6.5 down-regulation on the growth of transplanted tumors in vivo was verified by nude mice tumorigenesis assay. The Cancer Genome Atlas (TCGA) database and sequence-based RNA adenosine methylation site predictor (SRAMP) website were used to predict the enzymes and corresponding methylation sites. MazF digestion was chosen to validate the binding sites. RNAi technology was used to observe the changes in cell function after interfering with the expression of the corresponding genes of the modified enzymes. MeRIP-qPCR was used to detect the level of RP11-426A6.5 m6A cell line treated with actinomycin D was used to observe the stability of RP11-426A6.5. Results: RP11-426A6.5 methylation and expression levels were significantly higher in LSCC tissues than those in paracancerous tissues (methylation levels: 23.828±4.975 vs 20.280±3.607; expression levels: 1.197±0.314 vs 1.015±0.170, all P values<0.05). RP11-426A6.5 expression levels were closely correlated with T stage (T1-2: 1.081±0.298 vs T3-4: 1.306±0.292, χ2=5.35, P<0.05). The postoperative survival of patients with high RP11-426A6.5 expressions was significantly lower than that of patients with low RP11-426A6.5 expression (P=0.046). Assays in vitro and in vivo showed that the downregulation of RP11-426A6.5 significantly decreased the proliferation, migration, and invasion abilities of LSCC cells and the growth of transplanted tumors. The binding of methyltransferase-like 3 (METTL3), an m6A-modified enzyme, to the corresponding methylation site of RP11-426A6.5 enhanced its stability and mediated its regulation of malignant behaviors of LSCC cells. Conclusions: RP11-426A6.5 can regulate the malignant behaviors of LSCC cells, which is mediated by the m6A modification process involving in the methyltransferase METTL3.

High-risk human papillomavirus in Turkish patients with clinically suspicious cervical lesions analyzed by multiplex-PCR.

Human papillomavirus (HPV) infection is known to be the main cause of cervical cancer. This study aimed to determine the prevalence of high-risk HPV genotypes in smear specimens taken from women who had normal or abnormal cytology using a multiplex PCR method. The study included 270 women aged between 19 and 69 yr with or without suspicious cervical abnormalities. A Pap smear sample from each patient was cytologically examined, and HPV typing was performed using a multiplex fluorescent PCR method. Those who were high-risk HPV positive and had a normal or abnormal cytology were further evaluated by colposcopy and biopsy. The total HPV positivity was 43 per cent (116/270). HPV positivity in the patients with an abnormal cytology was 77 per cent (33/43), whereas it was only 37 per cent (83/227) in women with normal cytology, which showed a significant difference (P<0.05). HPV positivity was also related to the age group when all the subjects were considered (P<0.05), and the highest prevalence of HPV infection was in the 30-39 yr age group. High-risk HPV types 16, 18, 31, 35, 51 and 56 were more common in the normal cytology patients, whereas high-risk HPV types 16, 31, 35, 45, 58 and 68 were commonly found in the abnormal cytology patients. The determination of high-risk HPV genotypes in women with clinically suspicious cervical lesions should be conducted during an annual follow-up, irrespective of a normal or abnormal cytology by the age of 30 years or above.

CircBNC2 affects epithelial ovarian cancer progression through the miR-223-3p/ LARP4 axis.

Epithelial ovarian cancer (EOC) is one of the most serious cancer. Circular RNA BNC2 (circBNC2) expression was decreased in EOC tissues. However, the molecular mechanism of circBNC2 remains unknown. The expression of circBNC2, microRNA-223-3p (miR-223-3p), and La-related proteins 4 ( LARP4 ) were detected by quantitative real-time fluorescence PCR (qRT-PCR). A series of in-vitro experiments were designed to explore the function of circBNC2 in EOC cells and the regulatory mechanism between circBNC2 and miR-223-3p and LARP4 in EOC cells. Western blot examined the protein levels of Snail1, Slug, and LARP4 . The relationship between miR-223-3p and circBNC2 or LARP4 was verified by Dual-luciferase reporter assays. The xenotransplantation model was established to study the role of circBNC2 in vivo . The expression of circBNC2 and LARP4 was decreased in EOC tissues, while the expression of miR-223-3p was increased. CircBNC2 can sponge miR-223-3p, and LARP4 is the target of miR-223-3p. In-vitro complement experiments showed that overexpression of circBNC2 significantly decreased the malignant behavior of EOC, while co-transfection of miR-223-3p mimics partially upregulated this change. In addition, LARP4 knockdown increased the proliferation, migration, and invasion of EOC cells inhibited by miR-223-3p inhibitor. Mechanically, circBNC2 regulates LARP4 expression in EOC cells by spongy miR-223-3p. In addition, in-vivo studies have shown that overexpression of circBNC2 inhibits tumor growth. Overexpression of circBNC2 decreased proliferation, migration, and invasion of EOC cells by regulating the miR-223-3p/ LARP4 axis, suggesting that circBNC2/miR-223-3p/ LARP4 axis may be a potential regulatory mechanism for the treatment of EOC.

Getting rid of 'rain' and 'stars': Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg.

ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets ('stars'). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets ('stars') and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).

Exosomes Derived from Yak Follicular Fluid Increase 2-Hydroxyestradiol Secretion by Activating Autophagy in Cumulus Cells.

Exosomes in the follicular fluid can carry and transfer regulatory molecules to recipient cells, thus influencing their biological functions. However, the specific effects of yak follicular fluid exosomes on 2-hydroxyestrodiol (2-OHE2) secretion remain unknown. Here, we investigated whether yak follicular fluid exosomes can increase 2-OHE2 secretion through the activation of autophagy in cumulus cells (YCCs). In vitro cultured YCCs were treated with yak follicular fluid exosomes for 6, 12, and 24 h. The effects of yak follicular fluid exosomes on autophagy and 2-OHE2 secretion were evaluated through real-time quantitative fluorescence PCR (RT-qPCR), Western blotting (WB), transfected with RFP-GFP-LC3, immunohistochemistry, and ELISA. To further investigate whether 2-OHE2 secretion was related to autophagy, YCCs were administered with yak follicular fluid exosomes, 3-methyladenine (3-MA), and rapamycin (RAPA). The results revealed that treatment with yak follicular fluid exosomes activated autophagy in YCCs and increased 2-OHE2 secretion. Conversely, the inhibition of autophagy with 3-MA blocked these effects, suggesting that autophagy has an important role in 2-OHE2 secretion in YCCs. Treatment of YCCs with rapamycin showed similar results with yak follicular fluid exosomes as there was an increase in 2-OHE2 secretion due to the activation of autophagy in the treated cumulus cells. Our results demonstrate that autophagy is enhanced by yak follicular fluid exosomes, and this is associated with an increase in 2-OHE2 secretion in YCCs.

Acrobat: A Multicenter Prospective Study to Monitor Chimerism in Post Allogeneic Hemopoietic Cell Transplant Patients with AML, ALL, or MDS for Prediction of Engraftment and Relapse

BACKGROUND: Allogeneic hematopoietic cell transplantation (HCT) remains the treatment of choice for leukemia patients, yet its success is limited at least in part by disease relapse, which can be as high as 50% or more at two years post-HCT. While engraftment post-HCT is routinely measured by semi-quantitative fluorescent PCR of short tandem repeats (STRs), its low sensitivity (1-5%) limits its usefulness as a routine relapse monitoring tool. We have developed an ultra-sensitive, accurate, and precise microchimerism monitoring solution, AlloHeme that can potentially improve allogeneic HCT patient surveillance for engraftment and relapse. NGS-based AlloHeme can accurately measure microchimerism (mC, defined as <1% recipient DNA) with a limit of detection down to 0.035%. We hypothesized that detection of microchimerism and precise measurement of increasing mixed chimerism (iMC) using a highly sensitive test can predict disease relapse in patients post allo-HCT. OBJECTIVES: To study the role of mC and iMC measured by AlloHeme on relapse detection in patients with acute leukemias and myelodysplastic syndrome, we designed a prospective multicenter clinical trial - ACROBAT (Assessment of Chimerism in Recipients of Allogeneic Bone Marrow / Hematopoietic Cell Transplantation using AlloHeme Test). METHODS: The ACROBAT study will enroll approximately 260 allo-HCT recipients with AML, ALL and MDS across about 10 centers in the US. All patients will undergo serial chimerism monitoring in the first two years post-HCT. All AlloHeme testing will be conducted at CareDx's CLIA-certified/CAP-accredited laboratory. Chimerism will be monitored in the whole blood as well as for its constituent CD3+, CD15+ and CD33+ cellular subtypes. Bone marrow (whole bone marrow and CD34+ sub-type) chimerism will also be evaluated at specific timepoints. The study will determine the predictive value of mC and iMC for relapse detection and the lead time to relapse. Univariable and multivariable sub-distributional hazard regression models and joint models of longitudinal and time-to-event data will be used to determine the impact of mC and iMC measured at different time points on relapse post-HCT. A time-dependent ROC curve will be used to investigate the predictive ability of AlloHeme, MRD, and chimerism by STR for relapse detection. CONCLUSIONS: The ACROBAT study is a multicenter prospective clinical study designed to evaluate the role of increasing mixed chimerism and microchimerism for early relapse detection in patients who have undergone allo-HCT for AML, ALL or MDS.

Genetic characteristics of hantavirus detected in rodents in Shenzhen

Objective: To study the molecular epidemiological characteristics and genotypes of hantavirus carried by rodents in Shenzhen. Methods: Rodents were captured, and their lung samples were collected and grinded for RNA extraction. The hantavirus positive samples were classified by real-time fluorescence PCR. Rat lung nucleic acid samples were selected to amplify the nucleotide sequences of partial M fragments (G2 segment) and S fragments by reverse transcription-nested polymerase chain reaction (RT-nested PCR). The PCR products were then sequenced and homology and phylogenetic tree analyses were conducted. Results: A total of 200 rodents were captured, including 189 Rattus norvegicus, 9 Rattus flavipectus and 2 Mus musculus. The positive rate of hantavirus was 21.0% (42/200), all of the isolates were seoul virus (SEOV) strains. The positive rate of hantavirus in Bao'an district was highest (45.7%), and the difference in detection rate among districts were significant (χ2=25.60,P<0.05). A total of 25 G2 segment sequences and S fragment sequences of SEOV were obtained by virus gene sequencing, and their nucleotide homology was 95.3%-100.0% and 97.6%-100.0%, respectively. Compared with other reference sequences of S2 subtype, the nucleotide homology between the sample sequence and the reference sequence from Guangzhou was high. Analysis on nucleotide homology and phylogenetic tree showed that hantavirus carried by the rodents captured in Shenzhen belonged to SEOV S2 subtype. Analysis on amino acid variation sites revealed that there was a variation in the nucleocapsid protein encoded by S gene from Alanine to Threonine at the 973 position of BA-111. Conclusion: Hantavirus carried by rodents in Shenzhen belongs to S2 subtype of Seoul virus, which have little variation compared with the hantavirus strains obtained in other years in Shenzhen and surrounding provinces.

Modulation of the Functional State of Mouse Neutrophils by Selenium Nanoparticles In Vivo.

This study aimed to discover the immunomodulatory effect of selenium nanoparticles (SeNPs) on the functional state of neutrophils in vivo. Intraperitoneal injections of SeNPs (size 100 nm) 2.5 mg/kg/daily to BALB/c mice for a duration of 7-28 days led to the development of an inflammatory reaction, which was registered by a significant increase in the number of neutrophils released from the peritoneal cavity, as well as their activated state, without additional effects. At the same time, subcutaneous injections of the same SeNPs preparations at concentrations of 0.1, 0.5, and 2.5 mg/kg, on the contrary, modulated the functional state of neutrophils depending on the concentration and duration of SeNPs administration. With the use of fluorescence spectroscopy, chemiluminescence, biochemical methods, and PCR analysis, it was found that subcutaneous administration of SeNPs (0.1, 0.5, and 2.5 mg/kg) to mice for a short period of time (7-14 days) leads to modification of important neutrophil functions (adhesion, the number of migrating cells into the peritoneal cell cavity, ROS production, and NET formation). The obtained results indicated the immunostimulatory and antioxidant effects of SeNPs in vivo during short-term administration, while the most pronounced immunomodulatory effects of SeNPs were observed with the introduction of a low concentration of SeNPs (0.1 mg/kg). Increase in the administration time of SeNPs (0.1 mg/kg or 2.5 mg/kg) up to 28 days led to a decrease in the adhesive abilities of neutrophils and suppression of the expression of mRNA of adhesive molecules, as well as proteins involved in the generation of ROS, with the exception of NOX2; there was a tendency to suppress gene expression pro-inflammatory factors, which indicates the possible manifestation of immunosuppressive and anti-inflammatory effects of SeNPs during their long-term administration. Changes in the expression of selenoproteins also had features depending on the concentration and duration of the administered SeNPs. Selenoprotein P, selenoprotein M, selenoprotein S, selenoprotein K, and selenoprotein T were the most sensitive to the introduction of SeNPs into the mouse organism, which indicates their participation in maintaining the functional status of neutrophils, and possibly mediated the immunomodulatory effect of SeNPs.

Leptospira sanjuanensis sp. nov., a pathogenic species of the genus Leptospira isolated from soil in Puerto Rico.

Two spirochetes (designated strains LGVF01 and LGVF02T) were isolated from soil samples in San Juan, Puerto Rico in HAN media after selection using a combination of ELISA, agar plating, and colony screening by Fluorescent Antibody Testing (FAT) and PCR for lipL32 and secY. Isolates were helix-shaped, aerobic, fast-growing, and highly motile. Genome sequence analysis indicated that both strains should be classified as members of a novel species within the pathogenic (P1) clade of the genus Leptospira . The average nucleotide identity between the two strains was 99.2 %, but below 93.2 % when compared to any previously described leptospiral species. Serotyping of strain LGVF02T indicates that it does not belong within any serogroup of Leptospira suggesting it also represents a new serovar. Collectively, strains LGVF01 and LGVF02T represent a new species of pathogenic leptospires for which the name Leptospira sanjuanensis sp. nov. is proposed. The type strain is LGVF02T (=NVSL-LGVF02T=KIT0302T).

Prognostic value of lncRNAs related to fatty acid metabolism in lung adenocarcinoma and their correlation with tumor microenvironment based on bioinformatics analysis.

BackgroundAs a key regulator of metabolic pathways, long non-coding RNA (lncRNA) has received much attention for its relationship with reprogrammed fatty acid metabolism (FAM). This study aimed to investigate the role of the FAM-related lncRNAs in the prognostic management of patients with lung adenocarcinoma (LUAD) using bioinformatics analysis techniques.MethodsWe obtained LUAD-related transcriptomic data and clinical information from The Cancer Genome Atlas (TCGA) database. The lncRNA risk models associated with FMA were constructed by single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network (WGCNA), differential expression analysis, overlap analysis, and Cox regression analysis. Kaplan-Meier (K-M) and receiver operating characteristic (ROC) curves were utilized to assess the predictive validity of the risk model. Gene set variation analysis (GSVA) revealed molecular mechanisms associated with the risk model. ssGSEA and microenvironment cell populations-counter (MCP-counter) demonstrated the immune landscape of LUAD patients. The relationships between lncRNAs, miRNAs, and mRNAs were predicted by using LncBase v.2 and miRTarBase. The lncRNA-miRNA-mRNA regulatory network was visualized with Cytoscape v3.4.0. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using DAVID v6.8. Quantitative real-time fluorescence PCR (qRT-PCR) was performed to verify the expression levels of the prognostic lncRNAs.ResultsWe identified 249 differentially expressed FMA-related lncRNAs in TCGA-LUAD, six of which were used to construct a risk model with appreciable predictive power. GSVA results suggested that the risk model may be involved in regulating fatty acid synthesis/metabolism, gene repair, and immune/inflammatory responses in the LUAD process. Immune landscape analysis demonstrated a lower abundance of immune cells in the high-risk group of patients associated with poor prognosis. Moreover, we predicted 279 competing endogenous RNA (ceRNA) mechanisms for 6 prognostic lncRNAs with 39 miRNAs and 201 mRNAs. Functional enrichment analysis indicated that the ceRNA network may be involved in the process of LUAD by participating in genomic transcription, influencing the cell cycle, and regulating tissue and organogenesis. In vitro experiments showed that prognostic lncRNA CTA-384D8.35, lncRNA RP5-1059L7.1, and lncRNA Z83851.4 were significantly upregulated in LUAD primary tumor tissues, while lncRNA RP11-401P9.4, lncRNA CTA-384D8.35, and lncRNA RP11-259K15.2 were expressed at higher levels in paraneoplastic tissues.ConclusionIn summary, the prognostic factors identified in this study can be used as potential biomarkers for clinical applications. ceRNA network construction provides a new vision for the study of LUAD pathogenesis.

Adaptive Filtering Framework to Remove Nonspecific and Low-Efficiency Reactions in Multiplex Digital PCR Based on Sigmoidal Trends.

Real-time digital polymerase chain reaction (qdPCR) coupled with machine learning (ML) methods has shown the potential to unlock scientific breakthroughs, particularly in the field of molecular diagnostics for infectious diseases. One promising application of this emerging field explores single fluorescent channel PCR multiplex by extracting target-specific kinetic and thermodynamic information contained in amplification curves, also known as data-driven multiplexing. However, accurate target classification is compromised by the presence of undesired amplification events and not ideal reaction conditions. Therefore, here, we proposed a novel framework to identify and filter out nonspecific and low-efficient reactions from qdPCR data using outlier detection algorithms purely based on sigmoidal trends of amplification curves. As a proof-of-concept, this framework is implemented to improve the classification performance of the recently reported data-driven multiplexing method called amplification curve analysis (ACA), using available published data where the ACA is demonstrated to screen carbapenemase-producing organisms in clinical isolates. Furthermore, we developed a novel strategy, named adaptive mapping filter (AMF), to adjust the percentage of outliers removed according to the number of positive counts in qdPCR. From an overall total of 152,000 amplification events, 116,222 positive amplification reactions were evaluated before and after filtering by comparing against melting peak distribution, proving that abnormal amplification curves (outliers) are linked to shifted melting distribution or decreased PCR efficiency. The ACA was applied to assess classification performance before and after AMF, showing an improved sensitivity of 1.2% when using inliers compared to a decrement of 19.6% when using outliers (p-value < 0.0001), removing 53.5% of all wrong melting curves based only on the amplification shape. This work explores the correlation between the kinetics of amplification curves and the thermodynamics of melting curves, and it demonstrates that filtering out nonspecific or low-efficient reactions can significantly improve the classification accuracy for cutting-edge multiplexing methodologies.

Oxidative Stress Induces Bovine Endometrial Epithelial Cell Damage through Mitochondria-Dependent Pathways

Simple SummaryPolymorphonuclear neutrophil (PMN) count is the main diagnostic method of bovine endometritis. High neutrophil PMN counts in the endometrium of cows affected by endometritis suggest the involvement of oxidative stress among the causes of impaired fertility. The damage mechanism of oxidative stress on bovine endometrial epithelial cells (BEECs) is still unelucidated. The objective of this experiment was to investigate the relationship between oxidative stress and graded endometritis in dairy uteri and the molecular mechanism of oxidative stress injury to BEECs. Our research showed that there was an imbalance of antioxidant stress in dairy cow uterine with endometritis, oxidative stress damaged dairy cow endometrial epithelial cells through mitochondria-dependent pathways. These findings may provide new insight into the therapeutic target of bovine endometrial cell injury.Bovine endometritis is a mucosal inflammation that is characterized by sustained polymorphonuclear neutrophil (PMN) infiltration. Elevated PMN counts in the uterine discharge of dairy cows affected by endometritis suggest that oxidative stress may be among the causes of impaired fertility due to the condition. Nevertheless, the effects of oxidative stress-mediated endometritis in dairy cows largely remain uninvestigated. Therefore, fresh uterine tissue and uterine discharge samples were collected to diagnose the severity of endometritis according to the numbers of inflammatory cells in the samples. Twenty-six fresh uteri were classified into healthy, mild, moderate, and severe endometritis groups based on hematoxylin and eosin stain characteristics and the percentage of PMNs in discharge. BEECs were treated with graded concentrations of H2O2 from 50 μM to 200 μM in vitro as a model to explore the mechanism of oxidative stress during bovine graded endometritis. The expressions of antioxidant stress kinases were detected by quantitative fluorescence PCR to verify the oxidative stress level in uteri with endometritis. Reactive oxygen species were detected by fluorescence microscope, and inflammation-related mRNA expression increased significantly after H2O2 stimulation. Moreover, mRNA expression levels of antioxidant oxidative stress-related enzymes (glutathione peroxidase, superoxide dismutase, and catalase) and mitochondrial membrane potential both decreased. Further investigation revealed that expression of the apoptosis regulator Bcl-2/Bax decreased, whereas expression of the mitochondrial apoptosis-related proteins cytochrome c and caspase-3 increased in response to oxidative stress. Our results indicate that an imbalance exists between oxidation and antioxidation during bovine endometritis. Moreover, apoptosis induced in vitro by oxidative stress was characterized by mitochondrial damage in BEECs.

Skin Specimen as an Alternative to Brain Tissue for Post Mortem Rabies Diagnosis in Animals to Scale up Animal Rabies Surveillance

Background: Detection of the virus or some of its specific components using WHO and OIE recommended standard laboratory tests is the only way to get a reliable diagnosis of rabies. Brain tissue is the preferred specimen for post-mortem diagnosis of rabies in both humans and animals. Higher biosecurity requirements, skill and transportation facilities required for collection and transport of brain or whole carcass to the laboratory is one of the reasons for the poor rabies surveillance in animals. Point of care testing with simple, reliable and easy to operate devices would be an ideal approach for providing rapid results. Methods: The study evaluated diagnostic performance of two reference tests, DFAT and RTPCR on skin specimen, to assess its suitability as an alternative of brain tissue for post mortem rabies diagnosis in animals. Brain tissue and skin sample belonging to different species of animals (n=90) collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis. Results: Validation of RT-PCR on skin and DFAT on skin in comparison with DFAT on brain as gold standard gave a sensitivity of 98% (95% CI:94.1-100) and 80% (95%CI:71.8-88.2) respectively. Specificity was 100% in both tests. Conclusion: The findings highlight the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.

Integrated analysis of the expression profiles of the lncRNA-miRNA-mRNA ceRNA network in granulosa and cumulus cells from yak ovaries

BackgroundGrowing oocytes acquire the ability to mature through two-way communication between gametes and surrounding somatic cumulus cells (CCs). Granulosa cells (GCs) support oocyte growth, regulate meiosis progression, and modulate global oocyte transcription activity. However, the proliferation and differentiation of the yak ovary in GCs and CCs remain unclear. To characterize the important roles of long non-coding RNA, (lncRNA), microRNA (miRNA), and messenger RNA (mRNA), whole-transcriptome analysis was performed. Real-time quantitative fluorescence PCR was performed to verify the selected RNA sequences.ResultsImportant gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways related to differentiation and oocyte development were identified for the target genes of differentially expressed lncRNAs, miRNAs, and mRNAs. In total,6223 mRNAs (2197 upregulated, 4026 downregulated), 643 lncRNAs (204 upregulated, 479 downregulated), and 559 miRNAs (311 upregulated, 248 downregulated) were significantly altered between the two groups. Target genes involved in cell adhesion, cell differentiation, regulation of developmental processes, cell proliferation, embryo development, signal transduction, apoptosis, and aromatic compound biosynthetic processes were significantly enriched. These RNAs were involved in ECM-receptor interaction, MAPK signaling, Hippo signaling, PI3K-Akt signaling, cell cycle, cell adhesion, leukocyte trans-endothelial migration, and actin cytoskeleton regulation.ConclusionsA comprehensive analysis of the co-expression network of competing endogenous RNAs (ceRNAs) will facilitate the understanding of the process of granulosa cell proliferation and differentiation and offer a theoretical basis for the development of oocytes.

Long Noncoding RNA-5657 Inhibits the Proliferation of Pancreatic Cells and Insulin Secretion in Gestational Diabetes by Inhibiting the NADPH Oxidase 4 (NOX4)

Multiple comorbidities are observed during pregnancy and there are many challenges for pregnant women with a previous history of diabetes. Pancreatic β-cell dysfunction is associated with gestational diabetes mellitus (GDM). This study intends to explore LncRNA-5657’s effect on proapoptotic cell proliferation and insulin secretion in gestational diabetes. Real-time quantitative fluorescence PCR measured LncRNA-5657 expression in GDM patient placental tissues along with analysis of cell activity, proliferation and insulin secretion by MTT assay, BrdU staining and ELISA. The relation between LncRNA-5657 and NOX4 was assessed by dual luciferase reporter assay. LncRNA-5657 was overexpressed in placental tissue of GDM patients compared to normal pregnant women. LncRNA-5657 overexpression promoted cell proliferation and insulin secretion, which was reversed by LncRNA-5657 downregulation. In addition, LncRNA-5657 negatively regulated NOX4 expression. Overexpression of NOX4 abolished the effect of LncRNA-5657 mimics on pancreatic β-cells, and knockdown of LncRNA-5657 promoted insulin secretion by upregulating NOX4 expression. In conclusion, LncRNA-5657 inhibits insulin secretion and β-cell proliferation by targeting NOX4, indicating that LncRNA-5657 might be a novel target for the treatment of GDM.

Developing a multiplex PCR-based assay kit for bloodstream infection by analyzing genomic big data.

BackgroundIn recent years, the incidence of bloodstream infections (BSI) has increased, the composition of pathogenic bacteria has changed, and drug resistance among bacteria has gradually increased due to the widespread use of interventional techniques, broad‐spectrum antibacterial drugs, hormones, and immunosuppressive agents. Here, we have developed a multiplex PCR assay kit for the detection of pathogens (14 Gram‐negative bacteria, 15 Gram‐positive bacteria, and 4 fungi) in whole blood from patients with BSI using five‐color fluorescent multiplex PCR followed by capillary electrophoresis. Our assay exhibits a diagnosis of higher quality and an improved detection rate for common pathogens.MethodsA local genome DNA database of 33 pathogenic bacteria was constructed. Next, “Exhaustive” primer search of the full coding sequence of the reference genomes of these bacteria was performed. Panels with minimal interactions between primers and amplicons were selected by random sampling and testing by a recursive algorithm. Primers and Mg2+ concentrations and PCR reaction procedures were optimized to maximize the detection efficacy.ResultsThe LOD of the kit was determined as 100 copies/μl. Using clinical samples, results generated by this kit and regular blood culture method were found to be 95.08% consistent. Additionally, six pathogens which were unidentifiable by blood culture were successfully detected by this kit.ConclusionOur study provided a bioinformatics approach to the challenge of primer design in multiplex PCR, and combined with optimized wet lab practice, a multiplex PCR‐based assay kit for BSI with higher sensitivity and accuracy than blood culture was produced.

Patients with biallelic GGC repeat expansions in NOTCH2NLC exhibiting a typical neuronal intranuclear inclusion disease phenotype

We report two patients with autosomal dominant neuronal intranuclear inclusion disease (NIID) harboring the biallelic GGC repeat expansion in NOTCH2NLC to uncover the impact of repeat expansion zygosity on the clinical phenotype. The zygosity of the entire NOTCH2NLC GGC repeat expansion and DNA methylation were comprehensively evaluated using fluorescent amplicon length PCR (AL-PCR), Southern blotting and targeted long-read sequencing, and detailed genetic/epigenetic and clinical features were described. In AL-PCR, we could not recognize the wild-type allele in both patients. Targeted long-read sequencing revealed that one patient harbored a homozygous repeat expansion. The other patient harbored compound heterozygous repeat expansions. The GGC repeats and the nearest CpG island were hypomethylated in all expanded alleles in both patients. Both patients harboring the biallelic GGC repeat expansion showed a typical dementia-dominant NIID phenotype. In conclusion, the biallelic GGC repeat expansion in two typical NIID patients indicated that NOTCH2NLC-related diseases could be completely dominant.

Inconclusive results of SARS-CoV2 RT-qPCR: To retest or not?

Background: Reverse transcription-quantitative PCR (RT-qPCR) is widely used to detect SARS-CoV-2 infections. A small proportion (3-5%) of the samples turn out to be inconclusive which are difficult to interpret and require repeat testing. Methods: This study utilizing RT-qPCR for SARS-CoV-2 collected data from the viral RNA extracted using Maccura Mag-Bind RNA from NPOP specimen, then amplified and quantified using Maccura SARS-CoV-2 Fluorescent PCR kit. The data with inconclusive interpretation and re-test results were selected and further analysed. Results: The retrospective analysis of 247 inconclusive samples that were retested was included in the study. Among the inconclusive results from the first test, 80% of samples which expressed SARS-CoV-2 N and E genes (without ORF1ab gene) turned out to be positive in the repeat test (p &lt; 0.001), while 55% of samples that had only one gene expressed initially, were positive on repeat testing. The E gene was detected (without N and ORF1ab gene) in nine samples, of which seven were negative on re-testing. Conclusions: Our study suggests that it is beneficial to repeat the SARS-CoV-2 RT-qPCR test, especially when two genes are expressed, while detection of only E gene in the first test can be regarded as negative.

Pre-implantation genetic testing for Marfan syndrome using mini-sequencing

Marfan syndrome (MFS1) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. This study aimed to perform clinical PGT-M in a family with a history of MFS1 for two generations. A family with two members affected by MFS1 approached the hospital for PGT-M. The couple decided to join the project following extensive counselling and informed consent was obtained. The mutation contributory to MFS1 was identified using whole-exome sequencing (WES). A novel PGT-M protocol using multiplex fluorescent PCR and mini-sequencing was developed and tested. Ten blastocysts were subjected to PGT-M in two clinical PGT cycles. Mini-sequencing revealed four normal and six affected embryos. Microsatellite-based linkage analysis confirmed mutation analysis results in all samples. The embryos diagnosed as normal (non-MFS1) were chosen for transfer. A pregnancy was obtained in the third embryo transfer. Invasive prenatal diagnosis confirmed the normal genotype of the baby. This study demonstrated comprehensive management using the application of clinical-based diagnosis, WES for mutation identification within the MFS1 gene, mini-sequencing for embryo selection and microsatellite-based linkage analysis for backup of PGT-M results and contamination detection to assist couples in having a healthy child when there was a family history of Marfan syndrome. Impact Statement What is already known on this subject? Marfan syndrome (MFS1, OMIM#154700) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. Pre-implantation genetic testing (PGT) is an alternative to traditional invasive prenatal diagnosis (PND) giving the couples the chance of starting pregnancy with the confidence that the baby will be unaffected. Most of the previous PGT reports employed microsatellite-based linkage analysis. A few PGT studies used sequencing, mini-sequencing and mutation analysis; however, the details of the techniques were not described. What do the results of this study add? Single-cell PCR protocol using multiplex fluorescent PCR and mini-sequencing was developed and validated. Two clinical PGTs cycles for Marfan syndrome were performed. A healthy baby was resulted. The details of multiplex fluorescent PCR and mini-sequencing protocols are described in this study so that the procedures can be reproduced. What are the implications of these findings for clinical practice and/or further research? Embryo selection can help the family suffering from Marfan syndrome for two generations to start a pregnancy with confidence that their child will be unaffected. This study also shows the use of a mini-sequencing protocol for PGT, which can be a universal protocol for other mutations by changing the PCR primers and mini-sequencing primers.