•The COVID-19 Ag Respi-Strip assay is a new immunochromatographic diagnostic tool for antigenic diagnosis of SARS-CoV-2.•This test did not reduce significantly the number of samples outsourced for ...COVID-19 confirmation.•The sensitivity of this rapid test is poor and improvements are needed to enhance its performances.
The COVID-19 Ag (Antigen) Respi-Strip assay is a new immunochromatographic diagnostic tool recently available for antigenic diagnosis of SARS-CoV-2. The proposed sensitivity is not higher than 60 %, but its high specificity allows both quick decisions for the management of patients and confirmation by molecular diagnosis for only negative tests. However, from the first tests performed, we suspected that the sensitivity observed with routine use was much lower than that announced by the manufacturer.
Over a period of one month, we compared the negative results obtained with the COVID-19 Ag Respi-Strip kit with those obtained from qRT-PCR performed in a laboratory qualified for the molecular diagnosis of SARS-CoV-2. All samples tested were naso-pharyngeal smears from UTM-RT medium.
Of 774 patients tested, 714 negative samples were sent for confirmation, and 159 were found to be positive by qRT-PCR. The median positive percentage agreement was 23.9 % (95 % CI: 14.2 %–38.2 %). The Cohen’s kappa score was 0.35.
Using this immunochromatographic assay as a triage test did not significantly reduce the number of samples outsourced for COVID-19 confirmation by qRT-PCR. In addition, even if the turn-around time is short, the assay is completely manual, which is not suitable for large volumes of routine samples. The sensitivity of this rapid test is poor, and improvements are needed to enhance its performance.
Aims
To search for a set of reference genes for reliable gene expression analysis in the globally important marine coccolithophore Emiliania huxleyi–virus model system.
Methods and Results
Fifteen ...housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1‐2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) were evaluated for their stability as potential reference genes for qRT‐PCR using ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software. CDKA, TUA and TUB genes were tested as loading controls for Western blot in the same sample panel. Additionally, target genes associated with cell apoptosis, that is metacaspase genes, were applied to validate the selection of reference genes. The analysis results demonstrated that putative housekeeping genes exhibited significant variations in both mRNA and protein content during virus infection. After a comprehensive analysis with all the algorithms, CDKA and GAPDH were recommended as the most stable reference genes for E huxleyi virus (EhV) infection treatments. For Western blot, significant variation was seen for TUA and TUB, whereas CDKA was stably expressed, consistent with the results of qRT‐PCR.
Conclusions
CDKA and GAPDH are the best choice for gene and protein expression analysis than the other candidate reference genes under EhV infection conditions.
Significance and Impact of the Study
The stable internal control genes identified in this work will help to improve the accuracy and reliability of gene expression analysis and gain insight into complex E. huxleyi–EhV interaction regulatory networks.
•RT-PCR demonstrated that egr-miR-71 and egr-let-7 were specifically amplified in all the plasma samples from the infected individuals with hydatid cyst.•The results of this study showed that two ...hydatid cyst derived miRNAs, egr-miR-71 and egr-miR-let-7, were significantly down-regulated after three and six months’ post-surgery and remove the cyst.
Currently, cystic echinococcosis (CE) follow-up is a serious concern among surgeons. MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs which are present in human body fluids in a highly stable form. Recently, it is observed that Echinococcus granulosus expresses a large number of miRNAs in its developmental stages. The current study aimed at evaluating the capacity of parasitic miRNAs to serve as plasma biomarkers for hydatid cysts before and after CE surgery. Hydatidosis patients were identified using radiological and histopathological examinations. Following RNA extraction and cDNA synthesis, the expression levels of parasite-derived miRNAs including egr-miR-71 and egr-let-7 were quantitatively evaluated using real-time polymerase chain reaction (RT-PCR) in 30 hydatid cyst-infected individuals before surgery and an equal number of healthy controls. Then, three- and six-month follow-ups were performed after cystectomy. To analyze parasite-derived miRNAs, the relative fold change between uninfected and infected samples was determined and normalized to hsa-miR-16–5p as the housekeeping internal control. RT-PCR demonstrated that egr-miR-71 and egr-let-7 were specifically amplified in all the plasma samples from the infected individuals with hydatid cyst; yet they were significantly down-regulated at three and six months' post-surgery (P < 0.05). The egr-miR-71 had a higher level of expression in larval stage compared with egr-let-7. The results of the current study indicated that hydatid cyst-derived miRNAs including egr-miR-71 and egr-let-7 can be detected in human plasma. Considering the changes in the expression levels of these miRNAs after three and six months, it seems that these miRNAs, especially egr-miR-71, could serve as novel promising biomarkers for the early diagnosis and monitoring of hydatidosis.
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Pigeon pea (Cajanus cajan) is widely cultivated for its nutritional and medicinal value yet remains an orphan crop as productivity has not been improved because of a lack of genome and non‐coding ...genome information. Non‐coding RNAs, like miRNAs and long non‐coding RNAs (lncRNAs), are involved in regulation of growth, metabolism, development, and stress response, and have a critical role in post‐transcriptional gene regulation (PTGR).
We attempted to elucidate the roles of miRNAs and lncRNAs in pigeon pea through experimental validation of computationally predicted miRNAs and lncRNAs and targets of miRNAs on mRNAs.
We experimentally validated 20 miRNAs and 11 lncRNAs. We predicted cleavage sites of three miRNA targets: serine/threonine‐protein kinase, polygalacturonase, beta‐galactosidase. We identified 469 targets of 265 miRNAs and their functional annotations using computational methods. We built a miRNA‐mRNA‐lncRNA network model, with the miRNAs targeting both mRNAs and lncRNAs, to obtain information on the interplay of these three molecules.
A confirmed interaction through experimental validation was established between miRNA, namely cca‐miR1535a targeting the mRNA for beta‐galactosidase, as well as the lncRNA cca‐lnc‐020033. Our findings increase knowledge of the non‐coding genome of pigeon pea and their roles in PTGR and in improving agronomic traits of this pulse crop.
Studying miRNA‐lncRNA‐mRNA interactions to decipher non‐coding RNA mediated post‐transcriptional gene regulation in Cajanus cajan.
As the most important transcription factor in the brassinosteroid (BR) signal transduction pathway, BES1 not only affects growth and development of plants but also regulates stress resistance of ...crops.
The physicochemical properties, gene structure, cis‐acting elements and gene chip expression of apple BES1 transcription factors were analysed using bioinformatics, and expression of this gene family was analysed with qRT‐PCR.
There were 22 members of the apple BES1 transcription factors, distributed on eight chromosomes, divided into seven subtribes (I–VII), and the same subtribe contained the same basic motifs. Gene structure analysis showed that the number and position of exons differed, and there was no upstream and downstream structure. Analysis of cis‐acting elements indicated that BES1 transcription factors contain response elements for hormones and abiotic stress, as well as organ‐specific elements. Gene chip expression profile analysis revealed that expression patterns of BES1 transcription factors differed in different apple hybrids and different organs. In addition, expression of apple BES1 genes was higher in flowers, young fruits, mature fruits and leaves. qRT‐PCR demonstrated that expression of MdBES1 genes was highest 12 h after BR induction. At the same time, there were differences in expression in response to PEG, NaCl and MeJA.
This paper provides a theoretical basis for analysis of the biological function and stress resistance mechanism of BES1 transcription factors in apple.
The paper identified 22 MdBES1 and its expressions under different hormones and stresses, laying a foundation for plant growth studies
Mammography is extensively used for breast cancer screening but has high false-positive rates. Here, prospectively collected blood samples were used to identify circulating microRNA (miRNA) ...biomarkers to discriminate between malignant and benign breast lesions among women with abnormal mammograms. The Discovery cohort comprised 72 patients with breast cancer and 197 patients with benign breast lesions, while the Validation cohort had 73 and 196 cancer and benign cases, respectively. Absolute expression levels of 324 miRNAs were determined using RT-qPCR. miRNA biomarker panels were identified by: (1) determining differential expression between malignant and benign breast lesions, (2) focusing on top differentially expressed miRNAs, and (3) building panels from an unbiased search among all expressed miRNAs. Two-fold cross-validation incorporating a feature selection algorithm and logistic regression was performed. A six-miRNA biomarker panel identified by the third strategy, had an area under the curve (AUC) of 0.785 and 0.774 in the Discovery and Validation cohorts, respectively, and an AUC of 0.881 when differentiating between cases versus those with benign lesions or healthy individuals with normal mammograms. Biomarker panel scores increased with tumor size, stage and number of lymph nodes involved. Our work demonstrates that circulating miRNA signatures can potentially be used with mammography to differentiate between patients with malignant and benign breast lesions.
•The stability in vitro of reference genes in the stages of proliferation and differentiation of bovine preadipocytes during cells was studied systematically.•The stable reference genes in vitro of ...bovine preadipocytes during cells proliferation stage and differentiation stage are not completely consistent.•The stable in vitro reference genes of bovine preadipocytes during cells at the proliferation stage and differentiation stage are GAPDH and RPS15A、 RPLP0 and EIF3K, respectively.
The stability of internal reference genes is crucial to the reliability of gene expression results using real-time fluorescence quantitative PCR (qRT-PCR). Inappropriate reference genes may lead to inaccurate results or even wrong conclusions. This study aims to identify stable reference genes for analyzing the expression of proliferation-related and differentiation-inducing genes in bovine primary preadipocytes (BPPs) in vitro. In this study, the stability of 16 candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K, RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) for qRT-PCR at proliferation and differentiation stages of BPPs was investigated by three different algorithms (geNorm, NormFinder and BestKeeper). The expression of two marker genes, PCNA and LPL, was used to determine the validity of the candidate reference genes (RGs) at the proliferation and differentiation stages, respectively. The results showed that GAPDH and RPS15A were the most stable RGs in the proliferation of bovine primary preadipocyte, while PPIA was the least stable internal reference gene. RPLP0 and EIF3K were the most stable RGs in the differentiation induction of bovine primary preadipocyte, while GAPDH was the least stable internal reference gene. This study of RGs laid the foundation for subsequent research into the mechanism of proliferation and differentiation of BPPs in vitro using qRT-PCR.
•The TUB, AK, RPS15 are best candidate reference genes for developmental stages.•The RPS3 and GAPDH are best candidate reference genes for insecticide stress.•The EF1-α and TUB are preferential ...housekeeping genes under various conditions.
The quantitative real-time polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantitative analysis of gene expression. Normalization of gene expression to that of relatively stably expressed housekeeping genes is required to facilitate the study of gene expression and to obtain more accurate RT-PCR data. However, no studies of the stability of expression of housekeeping genes in Lymantria dispar have been reported. In the present study, BestKeeper, GeNorm and NormFinder statistical software was used to evaluate the expression of thirteen candidate reference genes in L. dispar under different conditions. The expression levels of candidate reference genes were determined for two biological factors (developmental stages and tissues) and four abiotic treatments (temperature, insecticide, CO2 and starvation). The results showed that the best candidate reference genes in L. dispar were TUB, AK, RPS15 for developmental stages, RPL32 and GAPDH for tissues, ACTB and EF1-α for CO2 stress, GAPDH and RPL32 for temperature stress, RPS3 and GAPDH for insecticide stress, and GAPDH and RPS3 for starvation stress. In summary, EF1-α and TUB are preferential housekeeping genes in L. dispar under various conditions. These results provide a basis for the further study of functional genes of L. dispar.
•Long-term ENF decreased the abundance of soil diazotrophs.•Long-term ENF decreased species richness and diversity of soil diazotrophs.•Soil pH and EC values were the main factors affecting soil ...diazotrophs.•The maize rhizosphere could enrich soil diazotrophs.•The diazotrophic bacterial variations under ENF might inhibit early maize growth.
Excessive nitrogen (N) fertilization (ENF) and low utilization efficiency of fertilizer-derived N in high-input, high-yield cropping systems are serious ecological and economic problems in China. To examine the effects of long-term ENF on soil diazotrophs in relation to maize growth, we performed a 5-year field trial whereby 0–300 kg N ha−1 were added to black soil under a maize monocropping system in Northeast China. At the end of the 5-year field trial, the abundance and community structure of diazotrophs in the bulk soil and rhizosphere were investigated at the maize jointing stage by real-time quantitative polymerase chain reaction (qRT-PCR) and high-throughput sequencing. The results showed that: 1) ENF (N300) limited maize growth in the early stages and did not increase grain yield compared with moderate N supplementation (N180); 2) ENF significantly reduced nifH copy number, species richness, and Shannon index values of soil diazotrophs by 53.7%–79.7%, 37.2%–47.6%, and 20.0%–31.6%, respectively, reduced the relative abundance of Burkholderia, and increased the relative abundance of Sphingobium; 3) ENF decreased soil pH and increased electrical conductivity, which were the main factors affecting soil diazotrophic community structure as determined by Mantel test; and 4) diazotrophs were 1.3–3.0 times more abundant in the rhizosphere than in bulk soil, but no differences in α-diversity and community composition of diazotrophs were observed between rhizosphere and bulk soil. Thus, long-term ENF has negative impacts on the soil diazotrophic community, inhibiting maize growth in the early stage. Split N fertilization or slow/controlled-released N fertilizer should be used to avoid the negative effects of ENF on the soil diazotrophic community structure and early maize growth.
Objectives
The aim of this study was to investigate cementogenesis and alveolar bone induction during in vivo periodontal tissue regeneration upon implantation of hTGF‐β3 in furcation defects of ...Papio ursinus and to evaluate the feasibility of gene expression studies.
Materials and Methods
Class II furcation defects (day 0) were prepared in mandibular first and second molars of three P. ursinus and on day 30 implanted with and without 75 μg hTGF‐β3 in Matrigel®matrix. On day 0, 30 and 90, cementum and alveolar bone were harvested for gene expression analyses. Coral‐derived bioreactors with and without 250 μg hTGF‐β3 were implanted in the rectus abdominis to monitor tissue induction.
Results
hTGF‐β3 induced cementogenesis with TGF‐β3, Cementum Protein‐1 (Cemp1) and Osteocalcin (OC) up‐regulation, and down‐regulation of BMP‐2 and OP‐1. Matrigel®matrix specimens showed up‐regulation of BMP‐2, TGF‐β3, and OC, with down‐regulation of OP‐1 and Cemp1. hTGF‐β3 induced alveolar bone with down‐regulation of OP‐1, TGF‐β3, OC, and Cemp1. hTGF‐β3 bioreactors induced bone at the periphery only. BMP‐3, BMP‐4, TGF‐β1 and TGF‐β3 were up‐regulated in the adjacent muscle with TGF‐β2 down‐regulation.
Conclusions
Cementogenesis and osteogenesis by hTGF‐β3 entail the expression and up‐regulation of TGF‐β3 and OC with fine tuning and modulation of BMP‐2 and OP‐1.
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