Quantitative Polymerase Chain Reaction Technology Research Articles

Systematic assessment of the reliability of quantitative PCR assays targeting IS900 for the detection of Mycobacterium avium ssp. paratuberculosis presence in animal and environmental samples.

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's Disease (JD), which is endemic to dairy cattle and also incriminated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. JD is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative polymerase chain reaction (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD disease. MAP IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry and TaqMan technology was used in 107 reports. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.

The endometrial cancer detection using non-invasive hypermethylation of CDO1 and CELF4 genes in women with postmenopausal bleeding in Northwest China.

The objective of this study was to verify the clinical predictive performance of methylated cysteine dioxygenase type 1 (CDO1m) and CUGBP Elav-like family member 4 (CELF4m) in endometrial cancer (EC) women with postmenopausal bleeding (PMB). A single-center, prospective, and case-control study was conducted in the Gansu Provincial Maternity and Child-care Hospital with 138 female postmenopausal patients enrolled in 2022. All patients underwent body mass index (BMI) detection, transvaginal ultrasonography (TVUS) detection, carbohydrate antigen 125 detection, and the cervical exfoliated cell CDO1/CELF4 gene methylation detection to analyze the sensitivity, specificity, and accuracy of different screening tests statistically with the biopsy and/or dilation and curettage (D&C) pathological diagnosis under hysteroscopy as the gold standard. There was no significant difference in age between the EC group and the non-EC group, P = 0.492. Using quantitative polymerase chain reaction (qPCR) technology, we validated the CDO1 and CELF4 methylation detection with 87.5% sensitivity and 95.9% specificity as a useful strategy for the triage of women with PMB for the detection of EC. In addition, 100% of type II EC (n = 6) were positively detected by the CDO1 or CELF4 methylation test. The CDO1 and CELF4 methylation test with high specificity as an auxiliary diagnostic tool or alternative method provides physicians with a reference to distinguish between benign and malignant tumors in women with postmenopausal bleeding, to justify the necessity of using invasive methods to confirm diagnosis.

Association of gene polymorphisms and the decreased expression of long non-coding RNA LOC553103 with rheumatoid arthritis.

Long non-coding RNAs (lncRNAs) are involved in many key bioprocesses, including the occurrence and development of rheumatoid arthritis (RA). We aimed to analyze the association of genetic variants of long non-coding RNA LOC553103 and its peripheral blood mononuclear cells (PBMC) expression with RA. We enrolled 457 RA patients and 551 healthy controls and conducted a case-control study to analyze the relationship between LOC553103 gene rs272879 and the susceptibility of RA by TaqMan single nucleotide polymorphism genotyping. Among them, we sampled 92 cases and 92 controls, respectively, to detect the PBMC level of LOC553103 using quantitative real-time polymerase chain reaction technology. We explored the association between LOC553103 rs272879 and its PBMC expression levels in 71 RA patients. Mann-Whitney, Chi-square, and Spearman correlation analysis were used for statistical analysis and P-value <.05 was considered statistically significant. The genotype frequency of LOC553103 rs272879 CC was increased, and CG was decreased in RA patients compared to the control group (χ2 = 6.772, P = .034). The LOC553103 expression level in PBMC of RA patients was downregulated compared to healthy control (Z = -4.497, P < .001). Moreover, negative correlations were observed between the PBMC level of LOC553103 and erythrocyte sedimentation rate (rs = -0.262, P = .018), white blood cell count (rs = -0.382, P = .004), platelet (rs = -0.293, P = .030), and disease activity score in 28 joints (rs = -0.271, P = .016) in RA patients. This study provides the first evidence supporting an association between LOC553103 gene polymorphisms and susceptibility of RA and a relationship of PBMC level of LOC553103 with clinical manifestations and laboratory indicators of RA patients.

Pasteurized Akkermansia muciniphila and its outer membrane protein Amuc_1100 alleviate alcoholic liver disease through modulating gut microbiota and host metabolism

Recent researches have reported that pasteurized Akkermansia muciniphila, as a paraprobiotic, still plays an active role in various metabolic diseases. However, its role in alcoholic liver disease (ALD) remains unexplored. This study aims to evaluate the impact and mechanisms of pasteurized A. muciniphila on ALD. Its effects on ALD were assessed by phenotypic, biochemical parameters, and histological features in a mouse model. Further investigations into underlying mechanisms were conducted through gut microbiome, metabolomics analyses and quantitative real-time polymerase chain reaction (qRT-PCR) technology. Heterologously expressed outer membrane protein Amuc_1100 was utilized to explore the molecular action mechanism. Our research demonstrated that pasteurized A. muciniphila can indeed alleviate alcohol-induced liver damage by modulation of gut microbiota and host metabolism. It suppressed the proliferation of pathogenic microbes and fostered the growth of probiotics. Additionally, it rectified alcohol-induced abnormalities in serum metabolic patterns, particularly in lipid metabolism. Specifically, pasteurized A. muciniphila elevated serum levels of 3-hydroxybutyric acid, palmitoleic acid, acetylcarnitine, DHA, and arachidonic acid in alcohol-fed mice. qRT-PCR results showed that it reversed the alcohol-induced upregulation of mRNA expression of hepatic fatty acid synthesis genes and decreased mRNA expression of fatty acid oxidation and transport genes. Importantly, we identified that Amuc_1100 could partially recapitulate the protective effects of pasteurized A. muciniphila on ALD, especially regarding hepatic lipid metabolism. This study enhances our comprehension of the function of pasteurized A. muciniphila and underscores its potential therapeutic application, along with Amuc_1100, in the treatment of ALD.

Correlation analysis of serum miR-145 and miR-210 with carotid artery stenosis and their predictive value for cerebral ischemic events

Objective To analyze the significance of serum miR-145 and miR-210 expression levels in the diagnosis of carotid artery stenosis. Methods During the same period, 55 healthy individuals who received physical examination in the same hospital were recruited as controls and assigned to a non-stenosis group. Among the included patients, there were 45 cases of mild stenosis, 14 cases of moderate stenosis, and 6 cases of severe stenosis after carotid color Doppler ultrasonography. The expression levels of miR-145 and miR-210 in serum were measured using real-time fluorescence quantitative polymerase chain reaction (qPCR) technology. Results The expression levels of serum miR-145 and miR-210 in carotid artery stenosis group were significantly lower than those in non-stenosis group (p < 0.001). Multivariate Logistic regression analysis showed that smoking history, diabetes, hypertension and total cholesterol were positively correlated with the occurrence of carotid artery stenosis (p < 0.05). The expression levels of miR-145 and miR-210 were significantly negatively correlated with carotid artery stenosis (p < 0.001). In addition, patients with carotid artery stenosis and low expression levels of miR-145 and miR-210 had a greater risk of cerebral ischemia (p < 0.05). Cox regression analysis showed that the low expression of miR-145 and miR-210 were independent predictors of cerebral ischemic events. ROC analysis confirmed that miR-145 and miR-210 had good diagnostic efficacy in cerebral ischemia (p < 0.001). Conclusion The decreased expression of miR-145 and miR-210 in serum is closely related to the diagnostic significance of carotid artery stenosis, and may be used to predict the occurrence of cerebral ischemic events.

Novel Variants of CEP152 in a Case of Compound-Heterozygous Inheritance of Epilepsy.

Introduction CEP152 encodes protein Cep152, which associates with centrosome function. The lack of Cep152 can cause centrosome duplication to fail. CEP152 mutates, causing several diseases such as Seckel syndrome-5 and primary microencephaly-9. Methods In this study, we reported a patient diagnosed with epilepsy in Tianjin Children's Hospital. We performed clinical examination and laboratory test, and whole-exome sequencing was performed for the proband's and his parents' peripheral blood. The suspected compound-heterozygous variant in the CEP152 gene was verified by Sanger sequencing and quantitative real-time polymerase chain reaction technology. Results We discovered three variants-two of them from CEP152 and one from HPD . The result showed the variants in CEP152 only. The patient presented with seizures frequently. Sanger sequencing showed two novel variants in CEP152 are in exon26 (NM_014985.3 c.3968C > A p.Ser1323*) and in exon16 (NM_014985.3 c.2034_2036del p.Tyr678*). Conclusions We reported a novel compound-heterozygous variant in the CEP152 gene in this study. Most of the phenotypes are Seckel syndrome and primary microencephaly, and the novel variant may cause an atypical phenotype that is epilepsy.

Effects of Copy Number Variations in the Plectin (PLEC) Gene on the Growth Traits and Meat Quality of Leizhou Black Goats

The plectin (PLEC) gene is crucial in regulating muscle development and maintaining the cytoskeleton. An abnormal expression of PLEC can lead to muscle atrophy and muscular dystrophy. In a previous study, we found that Leizhou black goats exhibit abundant structural variations in the PLEC gene. However, the genetic effects of these variations on growth traits and meat quality in goats are not fully understood. In this study, three PLEC copy number variations (CNVs) were identified in a population of 417 Leizhou black goats, using quantitative polymerase chain reaction (qPCR) technology. Population distribution analysis revealed a high abundance of various types of these three CNVs. PLEC mRNA was found to be highly expressed in muscle tissue and remained consistently high from 1 month to 24 months after birth. Specifically, the gain type of CNV-1 (chr14: 81056401-81064800) showed a significant association with PLEC mRNA expression in muscle tissue (p &lt; 0.01). The sequence of CNV-1 in PLEC shares similarities with three domain superfamilies associated with muscle development and skin disease. Furthermore, there were significant differences in chest circumference, body weight, carcass weight, the cross-sectional area of the longissimus dorsi lumbar muscle, and shear stress between different types of CNV-1 (p &lt; 0.05). Notably, goats with the CNV-1 gain type demonstrated better phenotypic values compared to those with loss and normal types. These findings suggest that PLEC CNV-1 could play a crucial role in the growth and muscle development of Leizhou black goats, making it a potential marker for assisted selection in goat breeding.

TMT quantitative proteomics reveals key proteins relevant to microRNA-1-mediated regulation in osteoarthritis

Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2, Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.

DeltaXpress (ΔXpress): a tool for mapping differentially correlated genes using single-cell qPCR data

BackgroundHigh-throughput experiments provide deep insight into the molecular biology of different species, but more tools need to be developed to handle this type of data. At the transcriptomics level, quantitative Polymerase Chain Reaction technology (qPCR) can be affordably adapted to produce high-throughput results through a single-cell approach. In addition to comparative expression profiles between groups, single-cell approaches allow us to evaluate and propose new dependency relationships among markers. However, this alternative has not been explored before for large-scale qPCR-based experiments.ResultsHerein, we present deltaXpress (ΔXpress), a web app for analyzing data from single-cell qPCR experiments using a combination of HTML and R programming languages in a friendly environment. This application uses cycle threshold (Ct) values and categorical information for each sample as input, allowing the best pair of housekeeping genes to be chosen to normalize the expression of target genes. ΔXpress emulates a bulk analysis by observing differentially expressed genes, but in addition, it allows the discovery of pairwise genes differentially correlated when comparing two experimental conditions. Researchers can download normalized data or use subsequent modules to map differentially correlated genes, perform conventional comparisons between experimental groups, obtain additional information about their genes (gene glossary), and generate ready-to-publication images (600 dots per inch).ConclusionsΔXpress web app is freely available to non-commercial users at https://alexismurillo.shinyapps.io/dXpress/ and can be used for different experiments in all technologies involving qPCR with at least one housekeeping region.

Highly expressed long non-coding RNA SNHG14 activated MSU-induced inflammatory response in acute gout arthritis through targeting miR-223-3p.

According to reports, long non-coding RNAs (lncRNAs) are involved in the regulation of many inflammatory diseases. Here, our main purpose was to ascertain the expression data of lncRNA SNHG14 in acute gouty arthritis (AGA) and to explore its possible mechanism in the regulation of AGA. Reverse transcription quantitative polymerase chain reaction technology was supplied to detect the lncRNA SNHG14 expression. A receiver operating characteristics curve was drawn to estimate the accuracy of lncRNA SNHG14 in AGA diagnosis. An in vitro AGA cell model was constructed by inducing THP-1 cells with monosodium urate (MSU). The concentrations of inflammatory factors such as interleukin-1β, interleukin-6, and tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay. The luciferase reporter gene was used to verify the relationship between lncRNA SNHG14 and miR-223-3p. In clinical analysis, the levels of serum lncRNA SNHG14 in AGA patients were significantly higher than those in the control group. Abnormally elevated lncRNA SNHG14 has high sensitivity and specificity for AGA diagnosis. In in vitro cell experiments, silencing lncRNA SNHG14 inhibited the inflammatory response of THP-1 cells stimulated by MSU, and the luciferase reporter gene proved that lncRNA SNHG14 could bind to miR-223-3p. In addition, the level of miR-223-3p declined in AGA patients and the AGA cell model. Overexpression of miR-223-3p is helpful to alleviate an MSU-induced inflammatory response. In the AGA cell model, lncRNA SNHG14, as an miR-223-3p sponge, induces a cellular inflammatory response by controlling the level of miR-223-3p, so aggravating the disease progress of AGA.

Does physical activity moderate the association between shorter leukocyte telomere length and incident coronary heart disease? Data from 54,180 UK Biobank participants.

Telomere shortening is a biological aging hallmark. The effect of short telomere length may be targeted by increased physical activity to reduce the risk of multiple aging-related diseases, including coronary heart disease (CHD). The objective was to assess the moderation effect of accelerometer-based physical activity (aPA) on the association between shorter leukocyte telomere length (LTL) relatively in the population sample and incident CHD. Data were from the UK Biobank participants with well-calibrated accelerometer data for at least 6.5days (n = 54,180). Relative mean LTL at baseline (5-6years prior to aPA assessment) was measured in T/S ratio, using a multiplex quantitative polymerase chain reaction (qPCR) technology, by comparing the amount of the telomere amplification product (T) to that of a single-copy gene (S). aPA measures included total number of events (at least 10-s continued physical activity > 32 milligravities [mg]), total volume, mean duration, mean intensity, and peak intensity of all events. LTL, aPA measures, and their interactions were associated with incident CHD (mean follow-up 6.8years) using Cox proportional hazards models adjusting for covariates. Longer LTL (relative to the sample distribution) was associated with reduced incidence of CHD (adjusted hazard ratio [aHR] = 0.94 per standard deviation [SD] increase in LTL, [95% CI, 0.90 to 0.99], P = .010). Incidence of CHD was reduced by higher total volume of aPA (aHR = 0.82 per SD increase in LTL, [95% CI, 0.71 to 0.95], P = .010) but increased by higher total number of events (aHR = 1.11 per SD increase in LTL, [95% CI, 1.02 to 1.21], P = .020) after controlling for other aPA measures and covariates. However, none of the interactions between LTL and aPA measures was statistically significant (P = .171).

Effects of grazing prohibition on nirK- and nirS-type denitrifier communities in salt marshes.

Grazing prohibition is an effective management practice to restore salt marsh functioning. However, the effects of grazing exclusion on denitrifying microbial communities and their controlling factors in salt marshes remain unclear. In this study, we surveyed soil physicochemical properties and above- and below-ground biomass and using quantitative polymerase chain reaction and Illumina MiSeq high-throughput sequencing technology to determine the relative abundance, composition, and diversity of nitrite reductase nirS- and nirK-type denitrifying bacterial communities associated with grazing prohibition treatments and elevations. The abundance of nirS-type denitrifiers increased with grazing prohibition time, whereas the abundance of nirK-type denitrifiers remained unaltered. Moreover, nirS-type denitrifiers were more abundant and diverse than nirK-type denitrifiers in all treatments. Grazing prohibition significantly altered the operational taxonomic unit richness, abundance-based coverage estimator, and Chao1 indices of the nirS-type denitrifying bacterial communities, whereas it only minimally affected the structure of the nirK-type denitrifying bacterial community. The results imply that the nirS community, rather than nirK, should be the first candidate for use as an indicator in the process of salt marsh restoration after grazing prohibition. Substances of concern, total nitrogen, and salinity were the key environmental factors affecting the abundance and community composition of nirS and nirK denitrifiers. The findings of this study provide novel insights into the influence of the length of grazing prohibition and elevation on nirS- and nirK-type denitrifying bacterial community composition in salt marshes.

A super sandstorm altered the abundance and composition of airborne bacteria in Beijing

Sandstorm, which injects generous newly emerging microbes into the atmosphere covering cities, adversely affects the air quality in built environments. However, few studies have examined the change of airborne bacteria during severe sandstorm events. In this work, we analyzed the airborne bacteria during one of the strongest sandstorms in East Asia on March 15th, 2021, which affected large areas of China and Mongolia. The characteristics of the sandstorm were compared with those of the subsequent clean and haze days. The composition of the bacterial community of air samples was investigated using quantitative polymerase chain reaction (qPCR) and high-throughput sequencing technology. During the sandstorm, the particulate matter (PM) concentration and bacterial richness were extremely high (PM2.5: 207 µg/m3; PM10: 1630 µg/m3; 5700 amplicon sequence variants/m3). In addition, the sandstorm brought 10 pathogenic bacterial genera to the atmosphere, posing a grave hazard to human health. As the sandstorm subsided, small bioaerosols (0.65–1.1 µm) with a similar bacterial community remained suspended in the atmosphere, bringing possible long-lasting health risks.

Effects of Probiotic Enterococcus faecium from Yak on the Intestinal Microflora and Metabolomics of Mice with Salmonella Infection.

Salmonella spp. are pathogenic bacteria that cause diarrhea, abortion, and death in yak and severely harm livestock breeding. Therefore, it is vital to identify a probiotic that effectively antagonizes Salmonella. To the best of our knowledge, few prior studies have investigated the efficacy of Enterococcus faecium against Salmonella. Here, we evaluated the enteroprotective mechanism of E. faecium in a mouse Salmonella infection model using hematoxylin-eosin (H&E) staining, quantitative real-time polymerase chain reaction (Q-PCR) technology, microbial diversity sequencing, and metabonomics. Enterococcus faecium inhibited the proinflammatory cytokines IL-1β, IL-6, TNF-α, and IFN-γ and promoted the anti-inflammatory cytokine IL-10. The Firmicutes/Bacteroidota (F/B) ratio and the abundances of Firmicutes and Akkermansia were significantly higher in the E. faecium than in the Salmonella group. Metabonomics and microbial diversity sequencing disclosed five different metabolites with variable importance in the projection (VIP) > 3 that were characteristic of both the Salmonella and E. faecium groups. Combined omics revealed that Lactobacillus and Bacteroides were negatively and positively correlated, respectively, with cholic acid, while Desulfovibrio was positively correlated with lipids in both the control and Salmonella groups. Desulfovibrio was also positively correlated with lipids in both the Salmonella and E. faecium groups. Enterococcus faecium antagonizes Salmonella by normalizing the abundance of the intestinal microorganisms and modulating their metabolic pathways. Hence, it may efficaciously protect the host intestine against Salmonella infection.

The Influence of MDR1 Expression Regulated by miR-138 through TRPS1 Signaling Pathway on Multidrug Resistance of Osteosarcoma and Formation of Bacterial Infection Biofilm.

This study was to investigate the effect of microribonucleic acid (mi-RNA) on the resistance of human multidrug resistance gene 1 (MDR1) to osteosarcoma through the Trico-nasal finger syndrome 1 (TRPS1) pathway, as well as the effect of mi-RNA on biofilm formation. For this purpose, firstly, the expression of MDR1 and TRPS1 in osteosarcoma cells was detected by quantitative polymerase chain reaction (qPCR) technology. Moreover, the clinical paraffin sections of osteosarcoma cells were collected to explore the correlation between MDR1 and TRPS1. Then, both the MG-38 cells expressing and not expressing miR-138 were expanded. Afterward, a plasmid with a full-length clone of the TRPS1 antibody was applied to transfect the cells. Besides, Q-OCR was employed to detect the expression of TRPS1 and MDR1, and the expression of TRPS1 protein and P-glycoprotein (P-gp) was detected by Western blot (WB). The MTT method was adopted to detect the changes in the median lethal dose of doxorubicin and cisplatin in cells from each group. The well plate was used to establish an in vitro bacterial infection biofilm model, and the above two transfected cells were added during the model establishment process. Moreover, the formation of biofilm in the two groups was observed. The result of the paraffin biopsy was 33% (25/75) of mi-RNA, the positive rate of TRPS1 was 18.6%, and the Pearson correlation coefficient of the two was 0.477. Under mi-RNA interference, the TRPS1 and MDR1 of the three system cells were sharply reduced, and the trend of changes between the two was the same. The tolerance of the mi-RNA interference group to doxorubicin, cisplatin, paclitaxel and 5-fluorouracil decreased steeply, and the median lethal dose dropped, while the non-mi-RNA interference group showed the opposite trend. In addition, the number of colonies in the interference group was less sharp than that of the control group and the non-mi-RNA interference group. The conclusion was that mi-RNA could control the expression of MDR1 through the TRPS1 pathway, thus affecting the multi-drug resistance of osteosarcoma and also influencing the formation of bacterial biofilms.

An environmental DNA assay for the detection of Critically Endangered angel sharks (Squatina spp.)

Abstract The three sympatric angel shark species occurring in the Mediterranean – Squatina squatina (the angelshark), Squatina aculeata (the sawback angelshark), and Squatina oculata (the smoothback angelshark) – are all classed as Critically Endangered on the International Union for Conservation of Nature (IUCN) Red List. There is a clear need to better quantify their current status, using appropriate non‐destructive methods, to help inform future conservation measures. This study introduces an environmental DNA (eDNA) assay able to detect and distinguish S. aculeata, S. oculata, and S. squatina in the Mediterranean Sea by combining probe‐based quantitative polymerase chain reaction (qPCR) technology and Sanger sequencing. The assay targets a 173‐bp barcode in the mitochondrial cytochrome c oxidase I (COI) gene. It was tested in silico, in vitro on tissue‐extracted DNA, and on eDNA extracted from filtration samples. This genus‐specific assay was applied to detect the presence of S. squatina in eDNA samples collected in Corsica, France. The target barcode was found in seven of 76 eDNA samples, revealing the presence of S. squatina in north‐western Corsica, where the shark has never been observed, and confirming its existence on the eastern coast. The study also demonstrates that using eDNA sampling, based on 30 L of seawater filtered close to the substrate with a waterproof peristaltic pump, it was possible to detect the eDNA of this rare benthic species. The results of detection can help identify critical areas for angel shark conservation and facilitate the development of local public awareness initiatives. This novel qPCR assay should be used for future applications in the Mediterranean and eastern Atlantic targeting angel sharks to better identify the remaining populations. In this study the qPCR assay was applied for S. squatina eDNA, but application to S. aculeata and S. oculata still needs to be validated in the field.

Combining conservation tillage with nitrogen fertilizer measures promotes maize straw decomposition by regulating microbial community and enzyme activities

Straw return can effectively improve the farmland soil microenvironment and soil fertility. However, excessive straw in the topsoil adversely affects seed germination and crop growth. At present, the characteristics and key driving factors of straw decomposition in dry farmlands are unclear. Based on the interaction between three tillage practices (i.e., zero-till (ZT) and chisel-till (CPT) vs. conventional plow-till (PT)) and three nitrogen fertilizer rates (i.e., low nitrogen (N1: 180 kg N ha-1) and high nitrogen (N3: 300 kg N ha-1) vs. normal nitrogen (N2: 240 kg N ha-1)), quantitative polymerase chain reaction (qPCR) technology and an enzymatic detection kit were used to investigate the effects of key straw-decomposition enzyme activities and microbial abundance on straw decomposition during the growth period of winter wheat. Between 2018 and 2020, repeated in situ measurements of straw decomposition indicated that zero-till and chisel-till significantly increased winter wheat yield (ZT: 10.94%; CPT: 12.79%) and straw decomposition velocity (ZT: 20%; CPT: 26.67%), compared with plow-till. N2 (240 kg N ha-1) significantly increased wheat yield (N1: 4.65%; N3: 5.31%) and straw decomposition velocity (N1: 26.33%; N3: 13.21%), compared to N1 (180 kg N ha-1) and N3 (300 kg N ha-1), respectively. The partial least squares pathway model showed significant positive direct effects of soil moisture, NO3-, NH4+, total nitrogen, bacteria, cellulase, laccase and xylanase activities on straw decomposition, while pH, fungi and Actinomycetes had significant negative direct effects on straw decomposition. Overall, conservation tillage (zero-till and chisel-till) combined with N2 (240 kg N ha-1) is beneficial for straw decomposition in the drylands of the Loess Plateau and improve straw resource utilization and basic soil fertility. The results of the study clarify the key drivers of straw decomposition in dryland farmlands, and provide new ideas for developing updated soil management practices and adaptive nitrogen application strategies to promote the resource utilization of straw and achieve the goals of carbon peaking and carbon neutrality.

Impact of HPV molecular testing with partial genotyping as a feasibility study in cervical cancer community screening program in South India.

Cervical cancer can be eradicated by 2030 by the implementation of a global strategy involving the vaccination of young girls against human papillomavirus (HPV), screening 70% of women in 30-69 years of age and treating 90% of the women with precancerous lesions. For a country with a large population like India, all the three strategies can be a challenge. There is a need for implementation of a high throughput technology that can be scalable. Cobas 4800, a multiplexed assay based on quantitative polymerase chain reaction technology, identifies HPV 16 and HPV 18 along with the concurrent detection of 12 pooled other high-risk HPV infections. This technology was used to test 10 375 women from the South Indian community for the first time as a feasibility program. Upon testing, high-risk HPV was found in 595 (5.73%) women. A total of 127 women (1.2%) were found to be infected with HPV 16, 36 women (0.34%) with HPV 18 and 382 women (3.68%) with the 12 pooled high-risk HPV and multiple mixed infections were found in 50 women (0.48%). It was observed that there was a high prevalence of high-risk HPV in younger women, 30-40 years of age and a second peak was observed at 46-50 years of age. The second peak had higher mixed infections in the 46-50 years of age and this association was statistically significant. We found that 24/50 (48%) of the multiple mixed high-risk HPV infections were in the age group 46-50 years. The current study is the first attempt from India, on a completely automated platform using Cobas 4800 HPV test in a community screening program. This study shows HPV 16 and HPV 18 infections, when differentiated, can be valuable for risk stratification in community screening program. Women in the perimenopausal age (46-50yrs) showed a higher prevalence of multiple mixed infections, signifying a higher risk.

Human papilloma virus infection and its associated risk for cervical lesions: a cross-sectional study in Putuo area of Shanghai, China

ObjectiveTo investigate the human papilloma virus (HPV) infection status, main subtypes and age distribution characteristics of women in the Putuo area of Shanghai.MethodsA total of 13,936 subjects were enrolled in this study. These subjects were 15–89 years old, with a mean age of 41.68. Real-time fluorescence quantitative polymerase chain reaction technology was used to detect 21 types of HPV.ResultsA total of 2,500 subjects with HPV infections were detected in 13,936 cervical exfoliated cell specimens (total infection rate 17.9%). There were 15 people aged below 20,486 people aged 21-30,876 people aged 31-40,484 people aged 41–50, 338 people aged 51–60, and 301 people aged > 60. In total, 1,893 (75.7%) subjects had a single type of HPV infection, 424 (16.9%) had a double infection, and 183 had triple or more infections (7.4%). The top 6 subtypes of HPV infection in the Shanghai Putuo District were HPV 52 (3.81%), HPV 58 (2.46%), HPV 16 (2.43%), HPV 53 (2.30%), HPV 81 (1.74%) and HPV 39 (1.5%). The number of high-risk HPV infections was 1,978, and the total infection rate was 14.19%. The number of intermediate-risk HPV infections was 578, and the total infection rate was 4.15%. The number of low-risk HPV infections was 338, and the total infection rate was 2.43%.ConclusionThe top 3 populations with HPV infection rates in the Putuo District, Shanghai, were ≤ 20 years old, older than 60, and 21–30 years old. The infection rate of HPV in cervical outpatient clinics was significantly higher than that of other departments. The 9-valent vaccine is recommended for HPV vaccination in this area.

Exosomes derived from M1 macrophages inhibit the proliferation of the A549 and H1299 lung cancer cell lines via the miRNA-let-7b-5p-GNG5 axis.

Almost all cells are capable of secreting exosomes (Exos) for intercellular communication and regulation. Therefore, Exos can be used as a natural therapeutic platform to regulate genes or deliver drugs to treat diseases. M1 macrophages inhibit tumor growth by releasing pro-inflammatory factors. This study explored the applicability of M1 macrophage exosomes (M1-Exos) as gene carriers and the effects on GNG5 protein, and further examined whether macrophage repolarization could inhibit tumor activity. M0 macrophages were polarized toward M1 using vitexin. Exos were obtained from M1 macrophages by ultra-centrifugation. The transwell non-contact co-culture system was used to co-culture M1 macrophages with HLF-α human lung epithelial cells or A549 or H1299 lung cancer cells. MTT, scratch, and transwell assays were used to detect the cell viability, migration, and invasion ability of cells in the four groups. Flow cytometry was used to detect the apoptosis rate of each group, and western blot (WB) analysis was performed to detect the change in the expression of proliferation- and apoptosis-related proteins. We screened the differentially expressed microRNAs using quantitative polymerase chain reaction technology. Luciferase reporter analysis was performed to explore the interaction between miRNA and protein. We used Xenografted A549 tumors in nude mice to study the effect of M1-Exos on tumor cell growth in vivo. The results showed that, under the M1 macrophage co-culture system, lung cancer cell viability, invasion, and migration ability decreased, and the number of apoptotic cells increased, will all indicators being statistically significant (P<0.05). The expression levels of PCNA, KI67, and Bcl-2 decreased significantly, but that of Bax increased (P<0.05). Exosomes can have the same effect on tumor cells as M1 macrophages. Exosomes can transport miR-let-7b-5p to tumor cells, and miR-let-7b-5p can inhibit tumor cell proliferation and promote tumor cell apoptosis by regulating the GNG5 protein level. M1-Exos inhibit the proliferation, invasion, and metastasis of lung cancer cells through miRNA-let-7b-5p and GNG5 signaling pathways and inhibit the anti-apoptotic ability of lung cancer cells.