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  • Zoledronate inhibits the differentiation potential of adipose-derived stem cells into osteoblasts in repairing jaw necrosis
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-23
    Xingzhou Qu; Zhen Wang; Kailiu Wu; Yang Wang; Liancheng Shan

    Objective To explore the inhibitory effects of zoledronate (ZOL) on adipose-derived stem cells (ADSCs) into osteoblasts for repairing jaw necrosis. Methods ADSCs were induced to differentiate into osteoblasts. The differentiation characteristics of osteoblasts was observed under inverted microscope by alizarin red staining. The transwell assay was performed to evaluate the migration of ADSCs co-cultured with osteoblasts and divided into ZOL group treated with ZOL and N-ZOL group without ZOL treatment. The differentiation and proliferation characteristics of ADSCs differentiated osteoblasts were observed respectively. The expression of CTSK (Cathepsin K) and FGFR3 (Fibroblast growth factor receptor 3) in osteoblasts were analyzed by immunofluorescence and western blot. Results The differentiation degree and proliferation of ADSCs to osteoblasts in N-ZOL group were both higher than those in ZOL group. The migratory cell number in ADSCs differentiation in ZOL group was higher than that of N-ZOL group. The protein expression of CTSK and FGFR3 in ADSCs differentiated to osteoblasts in ZOL group was higher than that in N-ZOL group. Conclusion The differentiation of ADSCs into osteoblasts is significantly inhibited by ZOL. Due to this reason, it may be difficult to achieve good results by ZOL induced ADSCs into osteoblasts in repairing jaw necrosis.

    更新日期:2020-01-23
  • Genetics of Parkinson's disease
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-21
    Christina M. Lill

    Almost two decades after the identification of SNCA as the first causative gene in Parkinson's disease (PD) and the subsequent understanding that genetic factors play a substantial role in PD development, our knowledge of the genetic architecture underlying this disease has vastly improved. Approximately 5–10% of patients suffer from a monogenic form of PD where autosomal dominant mutations in SNCA, LRRK2, and VPS35 and autosomal recessive mutations in PINK1, DJ-1, and Parkin cause the disease with high penetrance. Furthermore, recent whole-exome sequencing have described autosomal recessive DNAJC6 mutations in predominately atypical, but also cases with typical PD. In addition, several other genes have been linked to atypical Parkinsonian phenotypes. However, the vast majority of PD is genetically complex, i.e. it is caused by the combined action of common genetic variants in concert with environmental factors. By the application of genome-wide association studies, 26 PD risk loci have been established to date. Similar to other genetically complex diseases, these show only moderate effects on PD risk. Increasing this etiologic complexity, many of the involved genetic and environmental risk factors likely interact in an intricate fashion. This article aims to provide a comprehensive overview of the current knowledge in PD genetics.

    更新日期:2020-01-22
  • Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-21
    Hui-Hua Zheng; Shu-Jian Zhang; Jian-Tao Cui; Jia Zhang; Leyi Wang; Fang Liu; Hong-Ying Chen

    In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°Cfor CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/μL for CSFV and 36 copies/μL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

    更新日期:2020-01-21
  • Genome-wide analysis of mRNAs and lncRNAs in Mycoplasma bovis infected and non-infected bovine mammary gland tissues
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-21
    Selçuk Özdemir; Serdar Altun

    Mycoplasma bovis (M. bovis) causes diseases such as arthritis, pneumonia, abortion, and mastitis, leading to great losses in the bovine dairy industries. RNA types such as messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) play significant roles in regulating the immune responses triggered by bacteria. The expression profiles of mRNA and lncRNA as they occur in bovine mammary gland tissues infected with M. bovis are still not well understood. To illuminate this issue, transcription analysis of mRNA and LncRNAs were conducted on the mammary gland tissues belonging to Holstein cattle infected and not infected with M. bovis. The analysis revealed 1310 differentially expressed mRNAs and 57 differentially expressed lncRNAs in the bovine mammary gland tissues infected and not infected with M. bovis. In addition, 392 novel lncRNAs were detected, 19 of which were differentially expressed. Gene ontology analysis reveals that differentially expressed mRNAs and lncRNAs play significant roles in such vital biological pathways as metabolic pathways, T-cell receptor signaling, TGF-beta signaling, pathways in cancer, PI3K-Akt signaling, NF-kappa B signaling, mTOR signaling, and apoptosis, including in the immune response to cancer. Based on our literature review, this study is the first genome-wide lncRNA research conducted on bovine mammary gland tissues infected with M. bovis. Our results provide bovine mammary gland lncRNA and mRNA resources to understand their roles in the regulation of the immune response against the agent M. bovis in bovine mammary gland tissues.

    更新日期:2020-01-21
  • Exosomes derived from human bone marrow mesenchymal stem cells transfer miR-222-3p to suppress acute myeloid leukemia cell proliferation by targeting IRF2/INPP4B
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-20
    Feng Zhang; Yaqin Lu; Meng Wang; Junfeng Zhu; Jiajia Li; Pingping Zhang; Yuan Yuan; Fangbing Zhu

    Aim This study aims to explore the role and mechanism of exosomes derived from human bone marrow mesenchymal stem cells (hBM-MSCs-Exo) in regulating proliferation and apoptosis of acute myeloid leukemia (AML) cell line THP-1. Methods hBM-MSCs-Exo was isolated by ultra-centrifugation and administered into THP-1 cells to elucidate the effects of exosomes in THP-1 cells. Cell proliferation and apoptosis were examined by CCK-8 assay and flow cytometry, respectively. The expression of miR-222-3p, IRF2, and INPP4B were measured by qRT-PCR and western blot. The interaction between miR-222-3p and IRF2 was analyzed by luciferase reporter assay. Results Lower cell viability rate, higher apoptosis ratio, higher miR-222-3p expression, and lower IRF1/INPP4B expression were observed in THP-1 cells exposed to BM-MSCs-Exo. The proliferation-inhibitory and pro-apoptotic effects of BM-MSCs-Exo on THP-1 cells were markedly compromised when miR-222-3p expression in BM-MSCs-Exo was inhibited. Furthermore, miR-222-3p directly targeted IRF2 and negatively regulated IRF2/INPP4B signaling in THP-1 cells. Moreover, overexpression of either IRF2 or INPP4B counteracted the proliferation-inhibitory and pro-apoptotic effects mediated by BM-MSCs-Exo. Conclusion BM-MSCs delivered miR-222-3p via exosomes to inhibit cell proliferation and promote cell apoptosis by targeting IRF2 and negatively regulating IRF2/INPP4B signaling in THP-1 cells.

    更新日期:2020-01-21
  • Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-15
    Basem M. Ahmed; Haitham A. Amer; Jonas Kissenkoetter; Ahmed Abd El Wahed; Mahmoud M. Bayoumi; S. Böhlken-Fascher; Mahmoud A. Elgamal; Nahed Yehia; Ausama A. Yousif; Mohamed A. Shalaby

    Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Molecular diagnostic techniques like RT-PCR and real-time RT-PCR are considered the gold standard for identification of H5 influenza viruses in clinical samples. These techniques are hampered by the need of well-equipped laboratories, large space requirement, and relatively long time-to-result. Recombinase polymerase amplification (RPA) assay represents an excellent alternative to PCR since it is more simple, rapid, economic, and portable. Reverse transcription RPA (RT-RPA) assay was recently developed for sensitive and specific detection of H5N1 virus in 6–10 min. To ensure the accuracy of the developed assay, two approaches for using a positive control were evaluated in this study. These approaches included: 1) all-in-one (internal positive control; IPC), 2) two-tubes-per-one-sample (external positive control; EPC). Sigma virus (SIGV) RNA and turkey mitochondrial DNA were tested as positive controls in both approaches. For all-in-one approach, both targets (H5 and IPC) were strongly inhibited. In contrast, very good amplification signals were obtained for the two types of EPC with no effect on the analytical sensitivity and specificity of H5 RT-RPA assay in two-tubes-per-one-sample approach. The performance of EPC-based H5 RT-RPA was further validated using 13 tracheal swabs. The results were compared to real-time RT-PCR and proved superior specificity in detecting H5N1 but not H5N8 viruses. Inclusion of EPC did not affect the aptitude of both assays in terms of sensitivity, specificity and reproducibility. In conclusion, the two-tubes-per-one-sample approach was more reliable to control the false negative results in H5 RT-RPA assay.

    更新日期:2020-01-15
  • Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-15
    Kasanchi M. Momin; Arockiasamy Arun Prince Milton; Sandeep Ghatak; Shiny C. Thomas; Govindarajan Bhuvana Priya; Samir Das; Ingudam Shakuntala; Rajkumari Sanjukta; Kekungu-u Puro; Arnab Sen

    The polymerase spiral reaction (PSR), a novel isothermal method for targeted DNA amplification, was effectively applied to detect Salmonella in artificially spiked pork. The specificity of the developed PSR was tested using 16 Salmonella and 15 non-Salmonella strains. The PSR assay was 10-fold more sensitive than conventional end-point PCR, having a sensitivity comparable to real-time PCR. The limit of detection of the developed assay was 4 × 103 per gram of pork without enrichment and 4 CFU per gram after a 6 h enrichment. The detection of 4 CFU per gram of pork was achieved within 8 h. The PSR assay was successful, and accurate in comparison to microbiological methods, in detecting Salmonella in 11 of 76 commercial pork samples. Therefore the positive predictive value, negative predictive value and accuracy rate of the developed assay were 100%. Considering its rapidity, user-friendliness, simplicity, cost-effectiveness and equipment-free nature, this PSR assay is a promising tool for the food industry for the detection of Salmonella and prevention of Salmonella outbreaks and recalls.

    更新日期:2020-01-15
  • LncRNA AFAP1-AS1 promotes osteoblast differentiation of human aortic valve interstitial cells through regulating miR-155/SMAD5 axis AFAP1-AS1 promotes osteoblast differentiation of VICs
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-13
    Weilai He; Feng Li; Shibing Zhang; Zhengyan Zhu; Min Lin; Shenglin Ge; Ruyuan Zhou

    Aim Degenerative calcific aortic valve disease (DCAVD) is a common valve disease characterized by massive calcium deposits in the aortic valve. Osteoblast differentiation of valve interstitial cells (VICs) is responsible for the formation of calcific nodules. This study aims to explore the function and underlying mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 (actin filament-associated protein 1 antisense RNA 1) in the pathogenesis of DCAVD. Methods AFAP1-AS1, miR-155 and mRNA levels were detected by qRT-PCR. Protein levels were measured by Western blot. Calcification deposition was examined by Alizarin Red staining. The interaction between AFAP1-AS1 and miR-155, as well as miR-155 and SMAD5 was evaluated using luciferase reporter assay. Results AFAP1-AS1 expression was increased both in calcified aortic valves from DCAVD patients and after osteogenic induction in human VICs. Furthermore, AFAP1-AS1 overexpression promoted osteogenic differentiation of VICs, whereas AFAP1-AS1 knockdown inhibited osteogenic differentiation. Mechanistically, AFAP1-AS1 acted as a sponge for miR-155 to elevate SMAD5 expression. Further functional assays revealed that miR-155 mimic and SMAD5 silencing effectively reversed AFAP1-AS1-promoted osteogenic differentiation of VICs. Conclusion Collectively, AFAP1-AS1 promotes osteogenic differentiation of VICs, at least in part, by sponging miR-155 to upregulate SMAD5. This study sheds new light on lncRNA-directed therapeutics in DCAVD.

    更新日期:2020-01-14
  • Epitope mapping of the White Spot Syndrome Virus (WSSV) VP28 monoclonal antibody through combined in silico and in vitro analysis reveals the potential antibody binding site
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-11
    P.V. Shine; K.M. Shankar; B. Abhiman; N.S. Sudheer; R. Patil

    White Spot Syndrome Virus (WSSV) infecting shrimp is an enveloped double-stranded DNA virus. The WSSV is a member of the genus Whispovirus. The envelope protein VP28 is the most investigated protein of WSSV. In the present study, the epitope mapping of the monoclonal antibody (MAb) C-33 was carried out. Based on the epitope mapping results, an antigen-antibody interaction model was derived. Peptide scanning and confirmation of epitopes of MAb C-33 were carried out using the sequence data. The MAb was reactive to the epitope of both recombinant VP28 and the whole virus. The results of the study indicated the presence of an epitope region. The epitope region is found positioned within two peptides, covering 13 amino acids. Framework and CDR (complementarity determining regions) regions of heavy and light chain (VH & VL) sequences showed identity to germline immunoglobulin sequences. The Web Antibody Modelling (WAM) selected for further evaluation based on a comparative analysis of WAM and Rosetta server-generated models of the Fv region. The docking study using WAM generated model revealed that the residues from LEU 98 to GLY105 are active in antibody binding. The findings of this study could form a structural basis for further research in VP28 based diagnostics and therapeutics or vaccine discovery.

    更新日期:2020-01-13
  • A novel simple genotyping assay for detection of the ‘Gait keeper’ mutation in DMRT3 and allele frequencies in Azteca and Costa Rican Saddle Horse breeds
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-07
    Miguel Angel Ayala-Valdovinos; Jorge Galindo-García; David Sánchez-Chiprés; Theodor Duifhuis-Rivera; Rubén Anguiano-Estrella

    The ‘Gait keeper’ mutation in the DMRT3 gene alters locomotion and gait patterns in horses. This mutation (C>A) has been found in all gaited breeds of horses analyzed but is absent in most non-gaited breeds. We developed a new mutagenically separated polymerase chain reaction (MS-PCR) based method for simple detection of horse DMRT3 genotype. Our method was applied in a preliminary study to determine DMRT3 allele frequencies in 78 Azteca horses (AZ) and 53 Costa Rican Saddle Horses (CRSH). We found a wild-type C allele frequency of 100% in the AZ horses. For the CRSH, the wild-type C frequency and mutant A allele frequency were 88.7% and 11.3%, respectively.

    更新日期:2020-01-07
  • Detection of cyprinid herpesvirus 2 by loop-mediated isothermal amplification in combination with a lateral flow dipstick
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-03
    Yanli Li; Feng Lin; Lihui Sun; Aixia Huang; Jianming Chen; Guijie Hao; Xuemei Yuan; Haiqi Zhang; Shengqi Su

    We developed a convenient technique to detect Herpesviral haematopoietic necrosis attributed to cyprinid herpes virus 2 (CyHV-2), a serious disease of Crucian carp and goldfish related to high mortality. In the present study, we employed a lateral flow dipstick (LAMP-LFD) to present a loop-mediated isothermal amplification assay. The specificity was ascertained via other six viruses, and the sensitivity was compared using PCR method, which are the reaction conditions changes for the method improved. The results revealed that CyHV-2 performance was observable at 64 °C in a separated tube within 60 min, when the samples hybridized using an FITC-labeled probe. As the LAMP-LFD method's specificity was high, with its sensitivity identical to that of traditional PCR, the overall DNA collected revealed the lowest detection limit of 0.18 pg/μl from goldfish diseased by CyHV-2. In summary, the development of LAMP-LFD's method does not require expensive instruments, and it can be regarded as a fast, simple, and reliable method for CyHV-2 detection.

    更新日期:2020-01-04
  • Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-02
    Ying Wang; Ruhao Chen; Xianzhou Nie; Ziyang Zhong; Chunyan Li; Kun Li; Wei Huang; Xingyu Fu; Jun Liu; Bihua Nie

    In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the efficient and accurate detection of potato virus Y (PVY) under isothermal conditions. This RT-RPA assay was more efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay as the amplification reaction can be completed in less than 20 min. Moreover, unlike PCR that requires a thermocycler to carry out the DNA amplification through specific temperature phases, RPA assay could be performed under an isothermal condition at a temperature ranging from 25 to 40 °C. A simple instrumentation such as a heating block or a water bath or even anon-instrumental condition such as human hands or a benchtop inside/outside a room during the summer could satisfy the temperature requirement of RPA. The sensitivity of this assay was equivalent to that of the conventional RT-PCR, and the virus can be detected in a minimum of 2 pg of total RNA extracted from the PVY infected potato leaf tissues. The efficacy of the newly developed RT-RPA was then evaluated using field potato leaf and dormancy-broken sprout samples upon enzyme-linked immunosorbent assay (ELISA) screening. Of the 164 PVY-ELISA-positive samples, RT-RPA detected 157 whereas simplex RT-PCR detected 160 and multiplex RT-PCR detected 154. Of the 74 randomly selected PVY-ELISA-negative samples, RT-RPA, simplex RT-PCR and multiplex RT-PCR led to 1, 1 and 0 positive detections, receptively. Overall, RT-RPA and the two RT-PCR assays as well as ELISA exhibited an agreement of 96.6–98.7%, thus demonstrating the suitability of RT-RPA for large scale detection of PVY, irrespective of the strain type of the virus.

    更新日期:2020-01-02
  • Sp1 promotes dental pulp stem cell osteoblastic differentiation through regulating noggin
    Mol. Cell. Probes (IF 2.511) Pub Date : 2020-01-02
    Chun-peng Xia; Tao Pan; Nan Zhang; Jian-ran Guo; Bing-wu Yang; Di Zhang; Jun Li; Kai Xu; Zhen Meng; Hong He

    Based on the high self-renewal ability and osteoblastic differentiation capacity, dental pulp stem cells (DPSCs) are suggested to be promising cell source for osteogenesis. Therefore, illustrating the mechanism of osteoblastic differentiation of DPSCs is required. This current study aims to illustrate the role and mechanism of Sp1 in regulating osteoblastic differentiation of DPSCs. In this study, we downregulated Sp1 in DPSCs and evaluated the osteoblastic differentiation by measuring Runx2 and OCN expression with Western blot analysis and by Alizarin red staining. Furthermore, we investigated the mechanism of Sp1 regulating noggin with Firefly luciferase reporter gene assay and ChIP assay, and correspondingly evaluated the function of noggin in Sp1-regulated osteoblastic differentiation of DPSCs. We found that knockdown of Sp1 inhibits the expression of ALP, Runx2, COL1A1 and OCN, and decreases ALP staining, Alizarin red staining. Sp1 binds to noggin promoter and inhibits noggin expression, thus correspondingly regulates DPSCs osteoblastic differentiation. In conclusion, our study revealed that Sp1 regulates DPSCs osteoblastic differentiation through noggin and that Sp1/noggin can provide new perspective for enhancing DPSCs osteogenesis.

    更新日期:2020-01-02
  • Lung resided monocytic myeloid-derived suppressor cells contribute to premetastatic niche formation by enhancing MMP-9 expression
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-28
    Juechao Zhang; Xiaoqing Han; Huifang Shi; Yanyan Gao; Xuan Qiao; Huihan Li; Min Wei; Xianlu Zeng

    In cancer patients, the prevalence of myeloid-derived suppressor cells (MDSCs) is correlated with the degree of malignancy. In the present study, we investigated the role of circulating M-MDSCs in premetastatic niche formation using a mouse syngeneic tumor model and found that there was an increased frequency of M-MDSCs in the peripheral blood of tumor-bearing mice. M-MDSCs tracking and lung tissue histological analyses revealed that the malignant conditions promote the residence of circulating M-MDSCs and increased tumor cell arrest in the lungs. We further found that MMP-9 expression was increased in the circulating M-MDSCs and the administration of an MMP-9 inhibitor suppressed M-MDSCs transplantation-induced tumor cell arrest in the lung. Therefore, our findings suggest that the expansion of circulating M-MDSCs during tumor progression contributes to premetastatic niche formation by increasing MMP-9 expression.

    更新日期:2019-12-29
  • Multislice spiral CT images combined with CEA and lymphocyte-to-neutrophil ratio predict recurrence and post-operative metastasis of rectal cancer
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-28
    Hui-yuan Deng; Xiang-qing Zhu; Ying-ying Ding; Jin-dan Li; Jun Yang; Teng-fei Ke; Rui Wang; Qiang Chen; Jing Hu; Yan-ying Wang; Cheng-de Liao

    To explore the early predictors of post-operative recurrence and metastasis of rectal cancer, analyse the associated risk, and construct a model. Retrospective collection. Four hundred patients with rectal cancer underwent surgical resection and pathological diagnosis from September 2013 to September 2014. During the post-operative period, the patients were tested by imaging examination, serum tumour markers, and routine blood follow-up for at least 3 years. Preoperative CT examination of tumour size, lymphocyte-to-neutrophil ratio, and CEA were significant biomarkers for predicting recurrence and/or metastasis of post-operative rectal cancer. The stratified threshold of the lesion size cut-off point in CT images of patients with rectal cancer was 18.75 cm3, the cut-off point value of the lymphocyte-to-neutrophil ratio was 0.33, and the CEA cut-off point value was 16.97 ng/ml. We used the cut-off point to perform stratified survival analysis to obtain two K-M curves and conduct a log-rank test. The Cox multivariate risk regression results were as follows: preoperative CT images of lesion size, lymphocyte-to-neutrophil ratio, and CEA. The AUC of the normogram model for the prediction of post-operative recurrence and metastasis of rectal cancer is 0.939. Preoperative CT examination of tumour size can predict post-operative recurrence and metastasis of rectal cancer and can be used to analyse its risk. The lymphocyte-to-neutrophil ratio and CEA can also predict post-operative tumour recurrence and metastasis risk.

    更新日期:2019-12-29
  • Development of PCR based assays for detection of lethal Holstein haplotype 1, 3 and 4 in Holstein Friesian cattle
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-27
    Anshuman Kumar; I.D. Gupta; Govind Mohan; M.R. Vineeth; D. Ravi kumar; Jayakumar S; S.K. Niranjan

    Holstein haplotype (HH) 1, 3 and 4 are lethal mutations, responsible for early embryonic losses in Holstein Friesian (HF) cattle, worldwide. Three PCR based assays – tetra Amplification Refractory Mutation System PCR, PCR primer induced restriction analysis and PCR-restriction fragment length polymorphism techniques for screening of HH1, 3 and 4, respectively were developed and validated. During screening, six among 60 HF bulls were found as carrier for either of three mutations. These PCR assays are highly accurate and reproducible and can be used for screening of the haplotypes in HF cattle.

    更新日期:2019-12-27
  • lncRNA XIST attenuates hypoxia-induced H9c2 cardiomyocyte injury by targeting the miR-122-5p/FOXP2 axis
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-27
    Hui Peng; Yuxuan Luo; Yongjun Ying

    Objective To investigate the effect of lncRNA XIST on apoptosis induced by hypoxia. Methods We analyzed the expression levels of lncRNA XIST and miR-122-5p using RT-qPCR in hypoxia-induced cardiomyocytes. The mechanism by which lncRNA XIST affects myocardial ischemia was investigated using the cell transfection, CCK-8, and dual-luciferase reporter assays, as well as by flowcytometry, western blotting, and RNA immunoprecipitation. Results Hypoxic H9c2 cells demonstrated a decrease in their migration and invasion abilities and XIST expression and an increase in the extent of their apoptosis and expression of microRNA-122-5p. Overexpression of XIST significantly increased the H9c2 cell viability, enhanced cell migration and invasion, and decreased cell apoptosis in a hypoxic environment. The luciferase activity of XIST-WT in H9c2 cells co-transfected with XIST-WT and microRNA-122-5p mimics had decreased. The results of RNA immunoprecipitation showed that XIST interacted directly with miRNA-122-5p. Overexpression of XIST decreased the level of miRNA-122-5p significantly. mi-122-5p mimics increased H9c2 cell apoptosis and downregulated FOXP2 expression. Overexpression of FOXP2 upregulated the expression of the Bcl-2 protein in H9c2 cells transfected with microRNA-122-5p mimics and inhibited the expression of HIF-alpha, Bax, and the cleaved-caspase 9 protein. Conclusion lncRNA XIST could regulate the miR-122-5p/FOXP2 axis to attenuate hypoxia-induced H9c2 cardiomyocyte injury.

    更新日期:2019-12-27
  • Rapid analysis of Escherichia coli O157:H7 using isothermal recombinase polymerase amplification combined with triple-labeled nucleotide probes
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-27
    Jinqiang Hu; Yi Wang; Haijian Su; Huimin Ding; Xincheng Sun; Hui Gao; Yao Geng; Zhangcun Wang

    Rapid analytical methods are urgently needed to evaluate Escherichia coli (E. coli) O157:H7 in food. In this work, a novel recombinase polymerase amplification (RPA)-based lateral flow dipstick (LFD) method was developed to detect E. coli. Briefly, suitable primers and probes were designed and screened. Then, RPA reaction parameters, including volume, time, and temperature, were optimized. The specificity and sensitivity of RPA-LFD were analyzed, and a contaminated milk sample was used to test the detection performance of the proposed method. The optimal RPA reaction conditions included a minimum volume of 10 μL, incubation time of 10 min, temperature range of 39–42 °C, the primer pair EOF4/EOR3, and the probe EOProb. RPA-LFD was highly sensitive, it could detect as little as 1 fg of the genomic DNA of E. coli O157:H7, and 19 nontarget DNA of foodborne bacteria did not yield amplification products. Finally, the limit of detection of RPA-LFD for E. coli O157:H7 in artificially contaminated raw milk was 4.4 CFU/mL. In summary, the RPA-LFD assay developed in this study is an effective tool for the rapid investigation of E. coli O157:H7 contamination in raw milk samples.

    更新日期:2019-12-27
  • MiR-20a-5p suppressed TGF-β1-triggered apoptosis of human bronchial epithelial BEAS-2B cells by targeting STAT3
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-25
    Xiuyun Lv; Lihong Wang; Tianji Zhu

    Apoptosis of bronchial epithelial cells contributes to lung diseases, including asthma. Although miR-20a-5p is reportedly downregulated in the bronchial epithelia of asthmatic patients, its function and mechanism still need to be explored. Here, we explored how miR-20a-5p affects human bronchial epithelial cells stimulated with transforming growth factor (TGF)-β1. Using qRT-PCR, we observed downregulated miR-20a-5p levels in these cells. After transfecting miR-20a-5p mimics or inhibitors into human bronchial epithelium BEAS-2B cells, a Cell Counting Kit-8 assay and flow cytometry analysis showed that the mimics mitigated suppression of cell viability and acceleration of apoptosis that was triggered by TGF-β1, whereas the inhibitors exerted the opposite effects. TGF-β1 induced a decrease in expression of Bcl-2 and an increase in expression of Bax, both of which were inhibited by miR-20a-5p mimics and further enhanced by miR-20a-5p inhibitors. Further study verified that miR-20a-5p targeted the signal transducer and activator of transcription 3 (STAT3) and the STAT3 level was inversely related to the miR-20a-5p level. Furthermore, STAT3 overexpression partly counteracted the miR-20a-5p-induced anti-apoptotic effect in TGF-β1-treated BEAS-2B cells. In summary, this study suggested that miR-20a-5p restrained apoptosis in TGF-β1-stimulated BEAS-2B cells by targeting STAT3. MiR-20a-5p thus may be a novel therapeutic target for asthma treatment.

    更新日期:2019-12-26
  • MiR-202-5p attenuates neurological deficits and neuronal injury in MCAO model rats and OGD-induced injury in Neuro‐2a cells by targeting eIF4E-mediated induction of autophagy and inhibition of Akt/GSK-3β pathway
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-24
    Bing Li; Zhi Huang; Ju Meng; Wenfeng Yu; Hua Yang

    Ischemic stroke is a common cerebrovascular disease caused by insufficient blood supply to the brain. In recent years, studies have demonstrated that microRNAs (miRNAs) are involved in a variety of biological processes in the nervous system. However, the effects of miR-202-5p on cerebral ischemic stroke injury have not been completely elucidated. In our study, N2a cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, and middle cerebral artery occlusion (MCAO) rat models were constructed. Our results indicated that decreased miR-202-5p expression was connected to N2a cells after OGD/R-induced injury and rats after MCAO. In addition, high miR-202-5p expression increased proliferation and prevented apoptosis and autophagy of OGD/R-treated N2a cells, while also effectively decreasing the infarct volume in MCAO model rats. We validated the interplay between miR-202-5p and eukaryotic translation initiation factor 4E (eIF4E), and found that miR-202-5p downregulated eIF4E by targeted combination. Moreover, we demonstrated that miR-202-5p accelerated proliferation and suppressed autophagy of OGD/R-induced N2a cells by targeting eIF4E. Meanwhile, our other results suggest that upregulation of miR-202-5p may activate the Akt/GSK-3β pathway in ischemic brain injury. Our findings suggest that miR-202-5p may serve as a protective agent for ischemia-reperfusion injury in stroke via eIF4E.

    更新日期:2019-12-25
  • Origin recognition complex subunit 1 regulates cell growth and metastasis in glioma by altering activation of ERK and JNK signaling pathway
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-20
    Wenmin Xiong; Chen Xie; Yang Qiu; Ziwei Tu; Qiaoying Gong

    Origin recognition complex subunit 1(ORC1) is reported to be closely associated with the cell cycle. However, studies on the role of ORC1 in glioma remain undefined. The aim of the present study was to determine whether ORC1 affects cell migration, invasion, apoptosis, and proliferation and to explore the possible underlying mechanism. GEO database analysis indicated that ORC1 was significantly upregulated in glioma, while Gene set enrichment analysis (GSEA) analysis indicated that ORC1 primarily regulated the cell cycle and affects apoptotic signaling pathways. Analysis of protein-protein interaction (PPI) and gene ontology (GO) to further study the relevant mechanisms revealed that the function of the interaction between proteins and ORC1 was primarily concentrated in the regulation of cell cycle, and apoptosis played a critical role in the whole PPI network. Western blot assay and RT-PCR assay indicated that ORC1 was significantly upregulated in glioma tissues. Western blot assay and RT-PCR indicated that ORC1 was significantly upregulated in glioma cell lines. Cell migration, invasion, apoptosis, and proliferation were detected using Transwell and wound healing assays, flow cytometry, colony formation, and CCK8, respectively. Furthermore, OCR1 inhibition reduced invasion and migration, promoted cell apoptosis. In addition, OCR1 overexpression promoted cell proliferation and induced G2 phase arrest. Moreover, OCR1 downregulation suppressed activation of the ERK/JNK signaling pathway. The effects of ORC1 on biological processes were reversed by ERK and JNK inhibitors. These results indicate that ORC1 could be a novel prognostic marker of glioma via the activation of the ERK/JNK signaling pathway.

    更新日期:2019-12-20
  • Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-19
    Feng Cong; Fanwen Zeng; Miaoli Wu; Jingjing Wang; Bihong Huang; Yingying Wang; Qing Wang; Shouquan Zhang; Lei Ma; Pengju Guo; Weiwei Zeng

    Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.

    更新日期:2019-12-19
  • Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-09
    Xiujun Wu

    Background Chronic obstructive pulmonary disease (COPD) not only causes respiratory damage, but also affects circulatory function, which can be life-threatening in severe cases. Therefore, it is very essential to reveal the molecular mechanism of its pathogenesis. Methods Human Bronchial Epithelial cells were exposed to 20% cigarette smoke extract (CSE) condition to simulate the cells in COPD patients. GEO database was applied to analyze the expression of miR-380 in COPD patients. The expression level of miR-380 in CSE model was determined with qRT-PCR. Cells proliferation, apoptosis, and inflammation response-related factors were detected with cell counting kit 8, flow cytometry, and Western blot, respectively. A correlation between miR-380 and Cholinergic Receptor Nicotinic Alpha 4 subunit (CHRNA4) was predicted with bioinformatics software, and confirmed by dual luciferase assay. Rescue assay was applied to explore further relationship between miR-380 and CHRNA4. Results miR-380 showed a tendency of high expression in COPD patients and CSE models. Overexpression of miR-380 promoted the inhibitory effect of cells proliferation, and promotion effects of cells apoptosis and inflammation response, which were caused by CSE. CHRNA4, which was lower expressed in COPD patients, was affirmed as a target of miR-380 and negatively modulated by miR-380. Rescue assay indicated that exhausting of CHRNA4 attenuated the moderating effects of miR-380 inhibitor on cells damage induced by CSE. Conclusions Depletion of miR-380 alleviated cells damage caused by CSE through targeting CHRNA4, suggesting that miR-380/CHRNA4 may serve as novel therapeutic targets for COPD treatment.

    更新日期:2019-12-09
  • Establishment of canine macrophages stably expressing GFP-tagged canine LC3 protein for effectively detecting autophagy
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-07
    Dan Cui, Shanshan Huo, Xing Wang, Zhiqiang Zheng, Yonghong Zhang, Jianlou Zhang, Fei Zhong

    Autophagy plays a crucial role in eliminating protein aggregates, damaged organelles and invading pathogens. Genetically engineered cell line stably expressing green fluorescent protein (GFP)-tagged microtubule-associated protein light chain 3 (LC3) is extensively used to test autophagy through observing GFP puncta formation in the cells by fluorescence imaging. However, canine LC3 (cLC3) gene has not been cloned, therefore, GFP-tagged canine LC3 (GFP-cLC3) detection system has not been established. To generate GFP-cLC3 stably expressing canine-derived macrophages, the cLC3 cDNA was first amplified by RT-PCR and inserted into pEGFP-C1 plasmid to create GFP-cLC3 gene fusion. This genetic element was then transducted into canine macrophages mediated by lentivirus vector to generate the canine macrophages stably expressing fusion protein. Results showed that the sequence of cLC3 cloned in this study is highly homologous with other animals (80–95% homology). Phenotypic and functional analysis of these engineered cells revealed that GFP-cLC3 was indeed stably expressed and rapamycin or starvation can effectively induce GFP puncta formation in the cells, indicative of autophagosome formation. These GFP-cLC3-expressing cells may thus be useful to study autophagy in canine.

    更新日期:2019-12-07
  • HOXC10 promotes proliferation and attenuates lipid accumulation of sheep bone marrow mesenchymal stem cells
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-12-06
    Min Ma, Cuiru Wang, Yue Ao, Nimantana He, Fei Hao, Hao Liang, Dongjun Liu

    Homeodomain-containing gene C10 (HOXC10), known to regulate cell differentiation and proliferation, is a key negative regulator in the browning of white adipose tissue in mice. Sheep is an important farm animal that provides meat for human consumption, with fat content being an important meat quality determinant; however, there is no report about the role of HOXC10 in sheep adipocytes or adipogenesis. In this study, we investigated the effect of HOXC10 on proliferation and adipogenic differentiation in sheep bone marrow mesenchymal stem cells (sBMSCs). In sBMSCs, HOXC10 overexpression promoted cell proliferation and upregulated the expression of p-PI3K, p-AKT, p-p70S6K, p-MEK, and p-ERK, whereas HOXC10 knockdown was associated with the opposite effects. These results suggested that HOXC10 may promote cell proliferation by activating the MEK/ERK and PI3K/AKT/mTOR/p70S6K signaling pathways. In addition, we found that HOXC10 expression was negatively associated with lipid accumulation in adipogenic-differentiated sBMSCs. HOXC10 overexpression in sBMSCs significantly decreased lipid droplet accumulation and suppressed the expression of adipogenic-specific genes, including ACC, LPL, PPARG, and FABP4, while HOXC10 knockdown was associated with the opposite effects. Furthermore, our study suggested a new regulatory mechanism of the effect of HOXC10 on lipid accumulation and metabolism; HOXC10 may negatively regulate lipid accumulation in adipogenic-differentiated sBMSCs, at least in part, by suppressing LPL expression. Overall, our research not only contributes to a better understanding of the mechanism of lipid accumulation and metabolism in sheep, but also shed light on meat quality control in the future.

    更新日期:2019-12-06
  • lncRNA PVT1 aggravates doxorubicin-induced cardiomyocyte apoptosis by targeting the miR-187-3p/AGO1 axis
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-28
    Jiachen Zhan, Pengfei Hu, Yujie Wang

    Objectives To investigate the effect of long non-coding (lnc) RNA PVT1 on apoptosis induced by doxorubicin-induced cardiotoxicity. Methods We analyzed the expression levels of lncRNA PVT1, miR-187-3p, using reverse transcription real-time quantitative PCR (RT-qPCR) in doxorubicin-treated cardiomyocytes. The mechanism of lncRNA PVT1 in cardiotoxicity was investigated using cell transfection, CCK-8, flow cytometry, Western blot, and dual-luciferase reporter assays. Results Doxorubicin promotes H9c2 apoptosis and increased PVT1 expression in cardiomyocytes. Knockdown of PVT1 attenuated doxorubicin-induced cardiomyocyte apoptosis. We found that miR-187-3p is a direct target of PVT1, and that lncRNA PVT1 adsorbs miR-187-3p by sponge action, reducing miR-187-3p levels. miR-187-3p negatively regulates AGO1, and PVT1 regulates AGO1 expression by targeting miR-187-3p, thereby regulating apoptosis. In addition, we knocked down AGO1 in H9c2 cells transfected with the miR-187-3p inhibitor, and found that it inhibited apoptosis. Conclusion In doxorubicin-induced cardiomyocyte toxicity, the highly expressed lncRNA PVT1 enhances the expression of AGO1 by sponge adsorption of miR-187-3p. Decreasing the expression of lncRNA PVT1 inhibits the adsorption of miR-187-3p through competing endogenous (ce) RNA, thereby reducing the expression of AGO1 and decreasing the apoptosis of cardiomyocytes.

    更新日期:2019-11-29
  • Development of an EvaGreen based real-time RT-PCR assay for rapid detection, quantitation and diagnosis of goose calicivirus
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-17
    Su Lin, Shizhong Zhang, Shao Wang, Kaichun Xie, Dandan Jiang, Shifeng Xiao, Xiuqin Chen, Shaoying Chen

    An unclassified calicivirus (CV) detected in geese was recently reported and proposed as a new member of the family Caliciviridae. There is limited information about the epidemiology, etiology and detection method of goose-origin CV (GCV) to date. In this study, an EvaGreen based fluorescence quantitative real-time RT-PCR assay was developed and optimized for the detection of GCVs. The assay sensitively detected GCV RNA template with a good linear standard curve We also demonstrated the specificity and reproducibility of the detection method for GCVs. Thus, the method developed in this study will benefit the investigation of possible sporadic outbreaks of CV infections in geese, as well as epidemiological and etiological studies of GCVs.

    更新日期:2019-11-18
  • Verapamil and collagenase differentially affect collagen metabolism in experimental model of Peyronie's disease
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-13
    Jacek Karaszewski, Ilona Zareba, Tomasz Guszczyn, Barbara Darewicz, Jerzy Palka

    Objectives Peyronie's disease (PD) is accompanied by remodelling of connective tissue into fibrotic plaque. Treatment of the inflammatory and fibrotic phases of the disease is not established. The aim of the study was to evaluate the effect of verapamil (VER) and bacterial collagenase (COLL) on collagen metabolism and cell migration in fibroblasts with experimental wound healing and inflammation as an in vitro model of PD. Materials and methods In vitro model of PD was designed using experimental model of inflammation induced by Interleukin-1 (IL-1) in cultured fibroblasts and mechanical damage of the cells. Cell viability, cell proliferation, collagen biosynthesis, prolidase activity and cell migration were studied in both models of the cells treated with VER and COLL. Results VER decreased cell viability, DNA and collagen biosynthesis and increased prolidase activity in control fibroblast, while in “wounded” fibroblasts it significantly decreased all the processes. COLL did not affect cell viability and DNA biosynthesis, while inhibited collagen biosynthesis and prolidase activity in both control and “wounded” fibroblasts. In IL-1-treated fibroblasts VER inhibited all studied processes except prolidase activity, while COLL inhibited only collagen biosynthesis and prolidase activity. COLL accelerated cell migration, while VER attenuated the process in fibroblast model of wound healing, compared to control cells. Conclusion VER and COLL attenuate collagen biosynthesis in both fibroblast models. The VER-dependent inhibition of collagen biosynthesis was accompanied by inhibition of DNA biosynthesis at high prolidase activity, while COLL affected this process through inhibition of prolidase activity at high rate of DNA biosynthesis. It shows that anti-fibrotic activity of VER/COLL and anti-inflammatory activity of VER may represent approach to establish standard treatment of PD.

    更新日期:2019-11-13
  • Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-12
    Jinxing Song, Shanshan Zhao, Yueqin Li, Haiyan Wang, Liwei Zhang, Jun Wang, Yongshuai Peng

    Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species. We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described. The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 102 copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.

    更新日期:2019-11-13
  • Technical progress in circulating tumor DNA analysis using next generation sequencing
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-08
    Yunfei Bai, Zexin Wang, Zhiyu Liu, Geyu Liang, Wanjun Gu, Qinyu Ge

    Circulating tumor DNA (ctDNA) is tumor-derived, fragmented DNA that circulates freely in body fluids, predominantly in the peripheral blood. Recently, ctDNA analysis has been suggested as a complement to tissue biopsy in the detection and treatment of cancer. Genetic and epigenetic information specific to tumor cells, including single nucleotide variations, copy number variations, and modified methylation patterns, can be detected in ctDNA. Importantly, mutations in heterogenous tumors that could impart therapeutic resistance could be identified in ctDNA, which would aid in cancer diagnosis, prognosis, and real-time monitoring, and inform treatment with targeted therapies. However, ctDNA is still not a routinely used method for this purpose, because its detection techniques lack adequate sensitivity for reliable use in scientific studies and clinical trials. This review provides an up-to-date summary of ctDNA mutation detection methods based on next generation sequencing, highlighting their advantages and limitations, and focusing in particular on several optimized library preparation methods for improved sensitivity and specificity of ctDNA detection.

    更新日期:2019-11-08
  • MiRNA-483–5p is involved in the pathogenesis of osteoporosis by promoting osteoclast differentiation
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-06
    Keqian Li, Shenghao Chen, Pingyuan Cai, Kang Chen, Lei Li, Xu Yang, Jianhua Yi, Xingshun Luo, Yang Du, Hong Zheng

    Aims The study aimed to investigate the roles of miR-483–5p and IGF2 in osteoclast formation. Methods Blood and bone tissues were collected from osteoporosis and non-osteoporosis patients with hip fractures for gene expression analysis. CD14 + peripheral blood mononuclear cells (PBMCs) were isolated for differentiating osteoclasts. MiR-483–5p mimic and inhibitor was transfected into CD14 + PBMCs, respectively. Predicted by TargetScan and verified by Dual-luciferase reporter assay system, insulin-like growth factor-2 (IGF2) could be targeted by miR-483–5p. IGF2 expression vector was co-transfected with miR-483–5p mimic to study the role of IGF2 in miR-483–5p affecting osteoclast differentiation. Flow cytometry was performed for cell apoptosis analysis. Results High-expressed miR-483–5p and low-expressed IGF2 were frequently found in the serums and bone tissues derived from osteoporotic patients. We found that up-regulation of miR-483–5p in CD14 + PBMCs notably increased the number of TRAP-positive cells, at the same time, the expression levels of TRAP, nuclear factor of activated T-cells (NFATc1), cytoplasmic 1 (NFAT2) and Cathepsin K (CTSK) were also up-regulated. However, overexpressed IGF2 effectively reversed such effects produced by up-regulation of miR-483–5p on osteoclastogenesis-related factors in CD14 + PBMCs. Moreover, forced expression of IGF2 could also enhance apoptosis of osteoclasts reduced by miR-483–5p. Conclusions Our study suggests that miRNA-483–5p is involved in the pathogenesis of osteoporosis by promoting osteoclast differentiation.

    更新日期:2019-11-06
  • Rapid and quantitative detection of viable emetic Bacillus cereus by PMA-qPCR assay in milk
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-16
    Ping Zhou, Guoyang Xie, Taobo Liang, Bei Yu, Zoraida Aguilar, Hengyi Xu

    Emetic Bacillus cereus is one of the causative agents of foodborne diseases which can cause vomiting-type food poisoning after ingestion of contaminated food. To minimize B. cereus food poisoning, propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) called PMA-qPCR was applied for detecting viable emetic B. cereus in milk. The cereulide synthetase gene of emetic B. cereus (cesB) was chosen for the primer, and PMA treatment was optimized at 3 μg/mL to inhibit the PCR amplification of DNA from dead cells. Under optimized assay parameters, the limit of detection (LOD) using this method were 102 CFU/mL in both pure culture and in spiked milk matrix. The cycle threshold (Ct) values obtained for this assay was not significantly affected by the presence of non-target bacteria such as E. coli O157:H7 which indicated the high selectivity of the assay for emetic B. cereus. The PMA-qPCR assay used in this study has the potential for sensitive detection of viable emetic B. cereus in milk.

    更新日期:2019-11-04
  • A duplex PCR assay for the simultaneous detection and differentiation of Muscovy duck parvovirus and goose parvovirus
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-21
    Chunhe Wan, Longfei Cheng, Cuiteng Chen, Rongchang Liu, Shaohua Shi, Guanghua Fu, Hongmei Chen, Qiuling Fu, Yu Huang

    Both Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) can cause high mortality and morbidity in Muscovy ducklings. MDPVs and GPVs share high nucleotide identity, which can cause errors during differential diagnosis. In this study, the NS genes of both MDPVs and GPVs were chosen for the design of specific primers after genetic comparison. Only three primers (GF1, MF1 and MGR1) were designed for the duplex PCR assay: GF1 is specific for GPV only; MF1 is specific for MDPV only; and MGR1 is highly conserved for both MDPV and GPV. After a series of optimization experiments, the duplex PCR assay amplified a 161-bp fragment specifically for GPV, a 1197-bp fragment specifically for MDPV, and two fragments (161-bp and 1197-bp) for both GPV and MDPV. The lowest detection limit was 103 copies/μl. No amplification was obtained using nucleic acids from other pathogens (including DAdV-A, DuCV, DEV, GHPV, R.A., E. coli., P.M. and S.S.) occurring in Muscovy ducks. Application of the duplex PCR assay in field samples showed that even one-day-old Muscovy ducklings were both MDPV-positive and GPV-positive. In conclusion, a duplex PCR assay for the simultaneous detection and differentiation of MDPV and GPV was established using only three highly specific primers. Our finding suggested that country-wide vaccination with MDPV and GPV vaccines in waterfowls are necessary.

    更新日期:2019-11-04
  • A seminested RT-PCR for molecular genotyping of the Brazilian BR-I Infectious Bronchitis Virus Strain (GI-11)
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-07-28
    Ruy D. Chacón, Claudete S. Astolfi-Ferreira, Jorge L. Chacón, Luis F.N. Nuñez, David I. De la Torre, Antonio J. Piantino Ferreira

    Infectious bronchitis (IB) is one of the avian diseases with the greatest impact on poultry farming worldwide. In Brazil, strain BR-I (GI-11) is the most prevalent in poultry flocks. The present study aimed to develop a seminested RT-PCR assay specific for the diagnosis of BR-I IBV in Brazilian samples, targeting subunit 1 of the S gene. The detection limit of this assay was 10 copies of the IBV genome. In this study, 62.24% of 572 organ pools from the 5 regions of Brazil tested positive in a 3′UTR screening, and 84.83% were typed as BR-I IBV. BR-I was detected in the respiratory, digestive and urogenital tracts in pooled samples from all Brazilian geographical regions and in all the breeding systems analyzed. Specificity and sensitivity tests as well as phylogenetic analysis successfully confirmed the expected clustering of the sequences detected by this assay with the BR-I (GI-11) group. The nested PCR described in this study represents a suitable and valuable tool in the diagnosis, epidemiology, monitoring and vaccination decisions of IBV.

    更新日期:2019-11-04
  • Application of loop-mediated isothermal amplification (LAMP) for rapid detection of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus) and haddock (Melanogrammus aeglefinus)
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-07-08
    Yi Wang, Junli Feng, Xiaolan Tian

    Codfish is a commercially important species of sea fish and plays an important role in the world fishery. In our study, two loop-mediated isothermal amplification (LAMP) assays (real-time fluorescence LAMP and visual LAMP) were established for the identification of three cod species in Gadidae (Gadus morhua, Gadus macrocephalus and Melanogrammus aeglefinus). 12S rDNA gene was used to design primers to distinguish the Gadidae and non-Gadidae species, and the mitochondrial Cytb gene was selected for discrimination of three cod species. After optimization, the 12S rDNA system and species-specific systems performed well, and target cod DNA could be detected in single or mixed samples. In the species-specific systems, the absolute limit of detection (LODa) of three cod species were 285, 37 and 197 pg/μL, and the relative limit of detection (LODr) reached to 1%, 0.1% and 1%, respectively. In the 12S rDNA system, the LODa of three cod species were 28.5, 37 and 19.7 pg/μL, respectively, and the LODr reached to 0.1%. Through the detection of 13 commercial cod products, the LAMP systems can detect cod contents in raw materials and deep-processed products as well. It indicated that the methods developed in this study have strong practicability and can meet the needs of routine testing.

    更新日期:2019-11-04
  • Point-of-care detection of 16S rRNA of Staphylococcus aureus based on multiple biotin-labeled DNA probes
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-07-30
    Xiaoliang Zheng, Yue Wang, Shengjun Bu, Zhibao Chen, Jiayu Wan

    A visual method that combines multiple biotin-labeled DNA probes and lateral-flow nucleic acid biosensor was developed to detect Staphylococcus aureus. The 16S rRNA from Staphyloccocus aureus (S. aureus), coupled with multiple biotin-labeled DNA probes, was functionalized in a signal structure for lateral-flow point-of-care detection. The secondary structure of the 16S rRNA was unwound by two specific capture probes modified by Fam and multiple bridge probes, which extended additional sequences for use as initiators. By utilizing the initiators, each target 16S rRNA with multiple DNA probes could tether a number of biotin molecules, so that a large number of streptavidin-labeled gold nanoparticles could be introduced in the lateral flow assay. The images of the lateral flow detection results obtained using a smartphone were transmitted to a computer via Wi-Fi or Bluetooth connection for quantitative processing by ImageJ. The limit of detection was 103 cfu/mL without sample enrichment, and decreased to 0.12 cfu/mL following a 3-h enrichment of samples in growth medium. Notably, this method presented high specificity and applicability for the detection of S. aureus in food samples. In short, the developed visual non-specific operation method is very suitable for point-of-care diagnosis of pathogens in resource-limited countries.

    更新日期:2019-11-04
  • Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-12
    Shuo Jia, Baohua Feng, Zhuo Wang, Yingying Ma, Xuwen Gao, Yanping Jiang, Wen Cui, Xinyuan Qiao, Lijie Tang, Yijing Li, Li Wang, Yigang Xu

    Currently in China, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are the major causes of porcine viral diarrhea, and mixed infections in clinics are common, resulting in significant economic losses in pig industry. Here, a dual priming oligonucleotide (DPO)-based multiplex real-time SYBR Green RT-PCR assay were developed for accurately differentiating PEDV, TGEV, PoRV, and PDCoV in clinical specimens targeting the N gene of TGEV, PEDV, and PDCoV, and the VP7 gene of PoRV. Results showed that the DPO primer allowed a wider annealing temperature range (40–65 °C) and had a higher priming specificity compared to conventional primer, in which more than 3 nucleotides in the 3′- or 5′-segment of DPO primer mismatched with DNA template, PCR amplification efficiency would decrease substantially or extension would not proceed. DPO-based multiplex real-time RT-PCR method had analytical detection limit of 8.63 × 102 copies/μL, 1.92 × 102 copies/μL, 1.74 × 102 copies/μL, and 1.76 × 102 copies/μL for PEDV, TGEV, PoRV, and PDCoV in clinical specimens, respectively. A total of 672 clinical specimens of piglets with diarrheal symptoms were collected in Northeastern China from 2017 to 2018 followed by analysis using the assay, and epidemiological investigation results showed that PEDV, TGEV, PoRV, and PDCoV prevalence was 19.05%, 5.21%, 4.32%, and 3.87%, respectively. The assay developed in this study showed higher detection accuracy than conventional RT-PCR method, suggesting a useful tool for the accurate differentiation of the four major viruses causing porcine viral diarrhea in practice.

    更新日期:2019-11-04
  • Development of SYBR Green real-time PCR and nested RT-PCR for the detection of Potato Mop-top Virus (PMTV) and viral surveys in Progeny tubers derived from PMTV infected Potato tubers
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-15
    Hualan Zhou, Yan Lei, Pei Wang, Mingyue Liu, Xinxi Hu

    In this study, a new SYBR Green qPCR (qRT-PCR) and a nested RT-PCR (nRT-PCR) were developed to detect Potato mop-top virus (PMTV) in potato tuber tissues. The SYBR Green qRT-PCR and nRT-PCR assays were approximately 104- and 103- fold more sensitive than the conventional RT-PCR assay. The progeny tubers derived from PMTV-infected potato tubers were tested by conventional RT-PCR, SYBR Green qRT-PCR and nRT-PCR assays. Of the 17 samples, 9 (52.9%) were positive for PMTV by conventional RT-PCR, 11 (64.7%) were positive by nRT-PCR, and 17 (100%) were positive by SYBR Green qRT-PCR. Compared to nRT-PCR, SYBR Green qRT-PCR was showed to be more sensitive. The progeny plants exhibited foliar symptoms including chlorosis and reduction in leaf size when the PMTV-positive tubers were planted in a growth chamber at 20–22 °C. These findings suggest that PMTV has been passed on to the progeny plants and tubers.

    更新日期:2019-11-04
  • miR-34a and miR-29b as indicators for prognosis of treatment-free survival of chronic lymphocytic leukemia patients in Chinese Uygur and Han populations
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-16
    Yan Li, Min Mao, Hong Liu, Xiaomin Wang, Zhen Kou, Yuling Nie, Yichun Wang, Zengsheng Wang, Qin Huang, Tao Lang, Zailinuer Gu, Li An, Xiaoyan Zhang, Lin Fu

    The abnormal expression of miRNAs may play critical roles in the occurrence, development and prognosis of chronic lymphocytic leukemia (CLL), with potential ethnic differences being involved. p53 and immunoglobulin heavy chain variable region gene (IGVH) mutations were monitored and miRNA profile screening of CD19 + cells from Uygur CLL patients was performed, analyzed by miRNA arrays and verified using real-time PCR. There were 68 differentially expressed miRNAs in CD19 + B lymphocytes obtained from 6 Uygur CLL patients, of which miR-1295, miR-29b, miR-34a, miR-21 and miR-29c were the 5 most upregulated, and miR-181a, miR-126, miR-181b, miR-125a-5p and miR199b the 5 most downregulated miRNAs. miR-15a/miR-16-1 which might be important drivers of the disease, were not eliminated by profile screening. From the 68 differentially expressed miRNAs, 5 previously-reported CLL-related miRNAs were selected for further confirmation analyses, from which expression levels of miR-29b, miR-34a and miR-155 were found to be increased while miR-181a and miR-181b decreased. However, there were no differences in the expression levels of miR-15a/miR-16-1 between CLL patients and healthy donors, but the expression levels of miR-15a/miR-16-1 in CLL patients with a 13q deletion was depressed. In addition, there was no difference in the expression level of the above 7 miRNAs between 44 Han and 40 Uygur CLL patients. The expression levels of miR-29b, miR-181a and miR-181b correlated with IGVH mutations, while the expression levels of miR-34a, miR-29b and miR-181b correlated with a p53 abnormality in 84 Uygur and Han CLL patients. Taking p53 abnormality as the cut-off value criteria, low expression levels of miR-34a (cut-off value 4.65, P = 0.02) and miR-29b (cut-off value 4.71, P = 0.009) hinted at a poor treatment-free survival (TFS) prognosis for all CLL patients. Thus miR-34a and miR-29b may represent useful indicators for the prognosis of both Uygur and Han CLL patients.

    更新日期:2019-11-04
  • Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-08
    M.M. Gumaa, Xiaoan Cao, Zhaocai Li, Zhongzi Lou, Nianzhang Zhang, Zhijun Zhang, Jizhang Zhou, Baoquan Fu

    Brucellosis is a worldwide re-emerging zoonosis. It has an economic impact due to abortion and loss of fertility in livestock. In this study, Real-time recombinase polymerase amplification (RT-RPA-BP26) targeting Brucella spp. bp26 gene and Lateral flow dipstick (LFD-RPA-IS711) combined with SYBR- Green recombinase polymerase amplification (RPA) targeting insertion sequence IS711 region of Brucella spp. bp26 gene, was developed to detect Brucella spp. from different sample types in domestic animals. The sensitivity and specificity of the two developed RPAs were compared with real-time PCR, PCR, and Rose Bengal Plate Test (RBPT). The analytical sensitivity and detection limit of Real-time RPA and LFD RPA were four and six copies per reaction respectively. The detection of six colony forming units (CFU) of the bacteria-bearing construct with the target sequence was within 20 min at 40 °C for Real-time RPA and 37 °C for LFD RPA. The LFD RPA could work at temperatures between 30 and 35 °C and could be completed within 10–30 min. No significant differences were observed when comparing the results from Real-time RPA and LFD RPA to Real-time PCR and PCR. Both methods showed no cross reactivity with Chlamydia abortus, Toxoplasma gondii, Salmonella typhimurium, and Escherichia coli. In conclusion, RPA is a useful and convenient field and point of care test for brucellosis.

    更新日期:2019-11-04
  • Differential expression of miRNAs regulating NF-κB and STAT3 crosstalk during colitis-associated tumorigenesis
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-31
    Sherien M. El-Daly, Enayat A. Omara, Jihan Hussein, Eman R. Youness, Zakaria El-Khayat

    Inflammatory bowel disease (IBD) is mostly responsible for the development of colitis-associated colon cancer. Of the several signaling pathways involved in colonic inflammation, the activation and crosstalk between NF-κB and STAT3 serve as the pivotal regulatory hubs that regulate epithelial tumorigenesis by linking inflammation with cancer development. Understanding the molecular mechanisms regulating the crosstalk between NF-κB and STAT3 will help in targeting these signaling pathways and halt epithelial tumorigenesis. MicroRNAs (miRNAs) play important role in the regulation of NF-κB and STAT3 and function in a positive- or negative feedback loop to regulate the crosstalk of these transcription factor. In the present study we evaluated the aberrant expression of a selected panel of miRNAs (miR-181b, miR-31, miR-34a, miR-146b, miR-221, and miR-155) that regulate the crosstalk between NF-κB and STAT3 during colitis-associated tumorigenesis. We used the stepwise colorectal carcinogenesis murine model known as Azoxymethane (AOM)/Dextran sodium sulphate (DSS) to recapitulate the different stages of tumorigenesis. Our results revealed that the expression of the selected miRNAs changed dynamically in a stepwise pattern as colonic tissue transforms from normal to actively inflamed to neoplastic state, in accordance with the gradual activation of NF-κB and STAT3, suggesting that the aberrant expression of these miRNAs could function as the epigenetic switch between inflammation and colorectal tumorigenesis. We were able to elucidate the contribution of miRNAs in the NF-κB - STAT3 crosstalk during the stepwise development of colitis-associated carcinoma, and this could improve our understanding of the molecular pathology of colorectal tumorigenesis and even suggesting a therapeutic strategy by modulating the expression of these regulating miRNAs.

    更新日期:2019-11-04
  • PRPF4 is a novel therapeutic target for the treatment of breast cancer by influencing growth, migration, invasion, and apoptosis of breast cancer cells via p38 MAPK signaling pathway
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-08-22
    Song Park, Se-Hyeon Han, Hyeon-Gyeom Kim, Jain Jeong, Minjee Choi, Hee-Yeon Kim, Min-Gi Kim, Jin-Kyu Park, Jee Eun Han, Gil-Jae Cho, Myoung Ok Kim, Zae Young Ryoo, Seong-Kyoon Choi

    Pre-mRNA processing factor 4 (PRPF4), a core protein in U4/U6 snRNP, maintains snRNP structures by interacting with PRPF3 and cyclophilin H. Expression of the PRPF4 gene affects cell survival as well as apoptosis and is responsible for retinitis pigmentosa (RP). Proteomics analysis shows that PRPF4 may be a therapeutic target in human cancers. Nevertheless, the exact function and role of the PRPF4 gene are unclear. In this study, we assessed the expression of PRPF4 gene in human breast cancer cells. First, we confirmed that the PRPF4 gene was overexpressed in various breast cancer cell lines. Next, using breast cancer cell lines MCF7 and MDA-MB-468, we established stable cell lines with PRPF4 gene knockdown. We also performed microarray analysis to investigate molecular mechanisms underlying PRPF4 activity. All cell lines with PRPF4 gene knockdown exhibited reduced cell proliferation, remarkable reduction in anchorage-independent colony formation capacity, and reduction of PCNA protein, which is a marker cell of proliferation. Reduced expression of the PRPF4 gene induced apoptosis and changes in the expression of associated apoptotic markers in breast cancer cell lines. Knockdown of the PRPF4 gene reduced cellular capacity for migration and invasion (the key hallmarks of human cancers) and decreased the expression of genes involved in epithelial-mesenchymal transition (EMT). Microarray results showed that the expression of PPIP5K1, PPIPK2, and YWHAE genes was reduced at the transcriptional level, leading to reduced phosphorylation of p38 MAPK. These findings suggest that knockdown of PRPF4 gene slows down breast cancer progression via suppression of p38 MAPK phosphorylation. In conclusion, the PRPF4 gene plays an important role in the growth of breast cancer cells and is therefore a potential therapeutic target.

    更新日期:2019-11-04
  • Hsa-miR-346 plays a role in the development of sepsis by downregulating SMAD3 expression and is negatively regulated by lncRNA MALAT1
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-05
    Qiuhong Yang, Kaiqi Cao, Guangjun Jin, Jiancheng Zhang

    Sepsis is a common complication in infection, trauma, and surgery. Severe sepsis has been identified as the leading cause of death in patients suffering from noncardiovascular ailments in intensive care units. In the current study, we used lipopolysaccharide (LPS) to stimulate the mouse macrophage cell line RAW264.7, and investigated the effects of the lncRNA MALAT1/hsa-miR-346/SMAD3 regulatory network on the progression of sepsis. We showed that MALAT1 inhibited RAW264.7 cell proliferation, while hsa-miR-346 promoted its proliferation. In this RAW264.7 cell model, MALAT1 inhibited hsa-miR-346 expression, and upregulated SMAD3 protein expression. The SMAD3 protein expression in RAW264.7 cells was significantly downregulated upon the overexpression of hsa-miR-346. These results suggest that the MALAT1/hsa-miR-346/SMAD3 regulatory network plays a key role in the development of sepsis, and may serve as a target for the treatment of sepsis.

    更新日期:2019-11-04
  • Circ_016719 plays a critical role in neuron cell apoptosis induced by I/R via targeting miR-29c/Map2k6
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-04
    Chaogang Tang, Jianying Ou, Li Kou, Jinfeng Deng, Shijian Luo

    Background Stroke is a leading cause of mortality worldwide. Rac-MAPK kinase 6 (Map2k6) plays important roles in cell proliferation and apoptosis. However, the role played by Map2k6 in stroke injury and the underlying mechanism of action remain unknown. Methods Mice received cerebral ischemia/reperfusion (I/R) injuries by transient middle cerebral artery occlusion. HT22 cells were subjected to oxygen glucose deprivation and reoxygenation (OGD/R) to simulate an I/R injury. Subsequently, the levels of circ_016719, miR-29c and Map2k6 expression were determined, and their interactions were examined by luciferase assays. Circ_016719 knockdown, miR-29c inhibition or Map2k6 overexpression was induced in HT22 cells; after which, the cells were examined for their viability, apoptosis, autophagy and proliferation, as well their levels of Map2k6, p38, p53, LC3B–I, LC3B-II, Beclin 1, and p62 expression. Results Significantly increased levels of circ_016719 and Map2k6, and decreased levels of miR-29c were observed in both in vivo and in vitro I/R injury models. In HT22 cells, circ_016719 knockdown significantly increased miR-29c expression and cell proliferation, but decreased Map2k6 expression and cell apoptosis. Additionally, significant increases in LC3B–I and p62 levels and decreased LC3B-II levels were observed, indicating that circ_016719 knockdown had significantly inhibited autophagy. Furthermore, additional inhibition of miR-29c markedly suppressed the effects of circ_016719 knockdown; however, that suppression was significantly attenuated by Map2k6 overexpression. Additionally, Map2k6 was identified as a direct target of miR-29c, which in turn, might be sponged by circ_016719. Conclusions Our results suggest that circ_016719 directly targets miR-29c, and thereby regulates the expression and functions of Map2k6, which significantly contributes to the pro-apoptotic role of circ_016719.

    更新日期:2019-11-04
  • Identification of diarrheagenic Escherichia coli by a new multiplex PCR assay and capillary electrophoresis
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-11-01
    Jingyun Zhang, Yang Xu, Xia Ling, Yongming Zhou, Zheng Lin, Zheng Huang, Hongxia Guan, Yong Xiao, Wen Xu, Biao Kan

    Diarrheagenic Escherichia coli (DEC) is a set of the most common pathogens causing diarrhea. DEC strains are classified into five pathotypes based on the possession of different virulence genes: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) or Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). The development of an easy-to-use method to detect the specific virulence genes and distinguish the pathotypes is essential for the diagnosis and surveillance of DEC infections. In this study, a multiplex PCR assay (mPCR) specific to nine virulence genes and an internal control was designed for the identification of five DEC pathotypes. A temperature switch PCR (TSP) strategy was used in the PCR amplification. The PCR products were detected by capillary electrophoresis. The limit of detection (LOD) of the 10-plex reaction was 5 × 103 copies/reaction for stx2 and 5 × 102 copies/reaction for the other targets. The mPCR showed very high specificity, and inclusivity and exclusivity were both 100%. When the mPCR assay was used for the detection of 221 cryopreserved diarrhea specimens, DEC colonies were detected from 49 specimens, and the positive rate was 22.2%. The mPCR assay was sensitive and specific, and the amplified product could be analyzed easily. Thus, this method could be used effectively to identify the suspected colonies of DEC in the primary culture of the specimen.

    更新日期:2019-11-04
  • Development and evaluation of a Luminex xTAG assay for sulfonamide resistance genes in Escherichia coli and Salmonella isolates
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-10-31
    Fengjiao Xu, Fangui Min, Jing Wang, Yinzhu Luo, Shuwu Huang, Meiling Chen, Ruike Wu, Yu Zhang

    Clinically occurring sulfonamide resistance in gram-negative bacteria is codified by several sul genes, mostly associated with the mobilized genetic elements named integrons, and integrons are frequently found in plasmids. There are four sul genes (sul1, sul2, sul3 and sul4) that encode resistance to sulfonamides. The aim of the present study was to develop a bead-based xTAG assay for the simultaneous detection of all four sul genes and related Class 1 integrons (int1) in Escherichia coli and Salmonella isolates. The limits of detection ranged from 10 to 1000 copies/μL of input purified plasmid DNA. Forty-one bacterial isolates from clinical samples were examined using the newly developed xTAG assay and also by conventional PCR to determine the relative performance of each. The results obtained by xTAG assay showed higher detection rates and accuracy for sul genes than conventional PCR. It indicated that the xTAG-multiplex PCR is a convenient method for rapid identification of sul genes.

    更新日期:2019-11-04
  • Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-10-23
    Zhengwei Chen, Jun Huang, Fang Zhang, Yang Zhou, Huijie Huang

    Shrimp hemocyte iridescent virus (SHIV), which was first identified in white leg shrimp (Litopenaeus vannamei) in China in 2014, can cause extensive shrimp mortality and major economic losses in the shrimp farming industry in China. In this study, a novel real-time isothermal recombinase polymerase amplification (RPA) assay was developed using a TwistAmp exo kit for SHIV detection. First, five primers and a probe were designed for the major capsid protein gene (GenBank: KY681039.1) according to the TwistDx manual; next, the optimal primers were selected by a comparison experiment. The primers and probe were specific for SHIV and did not react with shrimp white spot syndrome virus (WSSV), shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp enterocytozoon hepatopenaei (EHP), and macrobrachium rosenbergii nodavirus (MrNV) samples, as well as pathogens of acute hepatopancreatic necrosis disease (AHPND). The RPA assay reached a detection limit of 11 copies per reaction according to probit regression analysis. In addition, RPA assay detected the positive plasmid samples at concentration of 1000 copies/μL within 16.04 ± 0.72 min at a single low operation temperature (39 °C). The results proved that the proposed RPA method was an accurate, sensitive, affordable, and rapid detection tool that can be suitably applied for the diagnosis of SHIV in field conditions and in resource-poor settings.

    更新日期:2019-11-04
  • Design and construction of COX-2 specific fluorescent probes
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-10-23
    Yi Chen

    Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays a key role in the inflammatory cascade through the generation of prostaglandins. Recent studies have found that COX-2 is closely related to the occurrence and development of tumors, angiogenesis and metastasis of tumors. Overexpressed COX-2 has become a predictive biomarker for progression of pre-malignant towards cancer in some tissues, which makes the detection of COX-2 of great value and clinical significance. This review highlights COX-2-specific fluorescent probes based on small organic molecules, including their design strategies and applications in the detection and imaging of COX-2 in vitro and in vivo. The progress suggests that fluorescence detection and imaging of COX-2 is a vital and rapidly technology for early diagnosis of tumors.

    更新日期:2019-11-04
  • Simultaneous detection of porcine reproductive and respiratory syndrome virus and porcine circovirus 3 by SYBR Green І-based duplex real-time PCR
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-10-23
    Lan-lan Zheng, Lv-ye Chai, Run-bo Tian, Yu Zhao, Hong-Ying Chen, Zhen-ya Wang

    The SYBR Green І-based duplex real-time PCR assay was developed for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 3 (PCV-3) genomes. PRRSV and PCV-3 were distinguished in the same sample by their distinctive melting temperature (Tm) which was 84 °C for PRRSV and 81.5 °C for PCV-3, and other non-targeted swine viruses showed no specific melting peaks. The detection limits of this assay were 46.1copies/μL for PRRSV and 49.3copies/μL for PCV-3, respectively. Thirty-three lung samples of porcine with respiratory and reproductive failure symptoms were collected and confirmed by the SYBR Green І-based real-time PCR assay and conventional PCR assay. The real-time PCR detection results showed that the PRRSV positive rate was 45.45%, the PCV-3 positive rate was 63.63%, the PRRSV and PCV-3 co-infection positive rate was 36.36%, which were more sensitive than conventional PCR detection. This duplex real-time PCR assay could be a rapid, sensitive and reliable method for the detection of PRRSV and PCV-3 co-infection.

    更新日期:2019-11-04
  • Utilization of recombinase polymerase amplification method combined with lateral flow dipstick for visual detection of respiratory syncytial virus
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-10-22
    Yu-zhong Xu, Du-zhi Fang, Fang-fang Chen, Qin-fei Zhao, Chao-ming Cai, Ming-gang Cheng

    Respiratory syncytial virus (RSV) is a major causative agent of respiratory tract infection necessitating hospitalization in children. A rapid diagnostic method would facilitate early detection of RSV infection and timely implementation of special treatment. Here, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with lateral flow dipstick (LFD) was evaluated for rapid visual detection of RSV. The primers were designed to target the conserved L gene. The RT-RPA-LFD assay could simultaneously detect RSV subtype A and B with the same detection limit of 10 copies of a given RNA molecule. Moreover, the assay showed no cross-reactivity with other common human pathogens. The performance of the RT-RPA-LFD assay was evaluated by testing 136 nasopharyngeal aspirates (NPAs). The agreement of the detection results between RT-RPA-LFD and qRT-PCR was 100% (34 positive and 102 negative cases). In summary, the developed RT-RPA-LFD assay had good performance in detecting RSV in clinical specimens, thus providing a novel alternative solution for the detection of RSV under field conditions.

    更新日期:2019-11-04
  • Ginsenoside Rb1 mitigates oxidative stress and apoptosis induced by methylglyoxal in SH-SY5Y cells via the PI3K/Akt pathway
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-10-16
    Fengwei Nan, Guibo Sun, Weijie Xie, Tianyuan Ye, Xiao Sun, Ping Zhou, Xi Dong, Jiafu Sun, Xiaobo Sun, Mengren Zhang

    Diabetic encephalopathy is a severe diabetic complication characterized by cognitive dysfunction and neuropsychiatric disability. Methylglyoxal (MGO), a highly reactive metabolite of hyperglycemia, serves as a major precursor of advanced glycation end products that play key roles in diabetic complications. Ginsenoside Rb1 (abbreviated as Rb1) has received extensive attention due to its potential therapeutic effects on diabetes and neurodegeneration. Therefore, this study aimed to investigate the effects of Rb1 on MGO-induced damage in SH-SY5Y cells and the related mechanism. SH-SY5Y cells were pretreated with Rb1 for 8 h and then exposed to MGO (0.5 mM) for 24 h. Cell survival was assessed by the MTT assay. Cell apoptosis was assessed using Hoechst 33342/propidium iodide (PI) staining and an Annexin-V/PI kit. The activities of oxidative stress markers were examined using commercial kits. Reactive oxygen species (ROS) staining and JC-1 staining were used to evaluate mitochondria injury. In addition, protein levels were measured by Western blot analysis. As a result, Rb1 alleviated the injury induced by MGO by increasing the activities of superoxide dismutase, catalase and total glutathione, decreasing the level of malondialdehyde, and alleviating mitochondrial damage and ROS production. Furthermore, Rb1 could enhance the Bcl-2/Bax ratio, inhibit the expression of cleaved caspase-3 and cleaved caspase-9, and enhance the levels of phosphorylated Akt. Moreover, the protective effects of Rb1 against MGO-induced apoptosis were partly abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. Our results demonstrated that Rb1 ameliorated MGO-induced oxidative stress and apoptosis in SH-SY5Y cells via activating the PI3K/Akt signaling pathway.

    更新日期:2019-11-04
  • miR-124 ameliorates depressive-like behavior by targeting STAT3 to regulate microglial activation
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-10-15
    Danning Lou, Jun Wang, Xiaohang Wang

    Major depressive disorder (MDD) is one of the most prevalent psychiatric disorders worldwide and a major public health concern that is associated with grave consequences. Systemic complexity and feedback processes among diverse drivers of the depression disorder contribute to the considerable variation in responses to the treatment of depression. Dysfunctional microRNA (miRNA) is involved in MDD. miR-124 is enriched in the brain and may be critical in neuronal differentiation. Previous studies have shown the value of miRNA-124 as a putative therapeutic target and a biomarker for major depression. However, the detailed mechanism of action of miR-124 in depression remains poorly understood. Here, we observed that miR-124 was downregulated in the hippocampus of mice with chronic unpredictable mild stress (CUMS). Restoration of miR-124 expression significantly attenuated depressive-like behavior and inhibited microglial activation induced by CUMS. Mechanistically, miR-124 directly targeted signal transducer and activator of transcription 3 (STAT3) in BV2 cells; in addition, upregulation of miR-124 inhibited the increase of inducible nitric oxide synthetase and proinflammatory cytokines, including IL-6, IL-1β, TNF-α, and MCP-1, in LPS-stimulated BV2 cells. The collective data suggest that dysfunction of miR-124 may be a foundation for the development of depression by promoting microglial activation.

    更新日期:2019-11-04
  • Rapid and sensitive real-time recombinase polymerase amplification for detection of Marek's disease virus
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-30
    Fanwen Zeng, Miaoli Wu, Lei Ma, Zongxi Han, Yue Shi, Yanping Zhang, Changjun Liu, Shouquan Zhang, Feng Cong, Shengwang Liu

    Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/μL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

    更新日期:2019-11-04
  • Development of HyBeacon® probes for the forensic detection of Panthera, rhinoceros, and pangolin species
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-26
    Kimberley George, Alice Masters, Nick Dawnay

    The Illegal Wildlife Trade (IWT) represents a multi-billion dollar black-market industry whereby wild species are illegally taken from their natural environment and sold. A common question asked by wildlife forensic scientists pertains to species and/or genus identity, which currently requires multi-step processing. Our work details the development of three HyBeacon® probes, used for the presumptive detection of rhinoceros, pangolin and key target species in the Panthera genus. The approach can be performed in a single tube using melt curve analysis and provide rapid assessment of sample identity. Using synthetic DNA of representative species, early data suggest the approach is sensitive enough to achieve species identification with <10 cells. Future development and assay validation can allow the rapid screening of multiple seized items before confirmatory DNA sequencing.

    更新日期:2019-11-04
  • Computational selection of minimum length groESL operon required for Anaplasma species attribution and strain diversity analysis
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-25
    Mourad Ben Said, Hanène Belkahia, Rachid Selmi, Lilia Messadi

    Anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, A. bovis, A. ovis and A. platys. Understanding the phylogenetic relations among these species is fundamental to perform an accurate identification and an informative intra-specific analysis. Heat shock groESL operon is frequently employed in phylogenetic analysis of Anaplasma species and, for the most cases, the use of partial sequences of this operon is randomly done without knowing the most appropriate regions to be used either in species attribution or in intra-specific diversity analysis. In this study, on the basis of all fully and nearly complete groESL sequences available in the GenBank, we firstly selected a minimum partial length sequence which allows species delineation and gives a similar topology to that found by analyzing the complete sequence. By using other in silico analyses, we obtained two minimal partial sequences that are the most interesting to describe intra-specific diversity within A. ovis and A. centrale. Our results raise concern on the use of randomly selected partial sequences of groESL operon employed for the detection and the characterization of Anaplasma species and provide additional background about minimum length groESL operon required for Anaplasma species attribution and strains diversity analysis.

    更新日期:2019-11-04
  • Comparative evaluation of loop-mediated isothermal amplification (LAMP) assay, GeneXpert MTB/Rif and multiplex PCR for the diagnosis of tubercular lymphadenitis in HIV-infected patients of North India
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-21
    N. Baikunje, D. Behera, A. Rajwanshi, M. Sharma, A. Sharma, K. Sharma

    Background Tubercular lymphadenitis (TBLA) is one of the most common extrapulmonary manifestations of tuberculosis in patients with HIV. With several other pathological conditions presenting as lymphadenitis and lack of consensus regarding a gold standard test, the diagnosis of TBLA remains a challenge for the clinician. Objectives and design: In this study, we have assessed the potential of loop-mediated isothermal amplification (LAMP) test for the diagnosis of TBLA in HIV-infected patients. The study group included samples collected by fine needle aspiration (FNAC) of lymph nodes from 24 HIV-infected patients with TBLA. A composite reference standard was used to identify cases of TBLA based on clinical suspicion, results of cytology, AFB smear, MGIT culture, GeneXpert MTB/RIF, multiplex polymerase chain reaction (MPCR) and subsequently clinical response to antitubercular therapy. These tests were also carried out in 26 control samples of lymph node FNAC from HIV-infected patients with non-tubercular lymphadenitis. Results LAMP assay was positive in 19/24 TBLA cases and yielded a sensitivity of 79.17% with 100% specificity. Cytology was suggestive in 18/24 (75%) TBLA cases. GeneXpert MTB/RIF assay correctly identified 16/24 TBLA cases, but the test did show one false positive result reducing its specificity. MPCR had the highest sensitivity of 91.67% as it correctly identified 22/24 cases and showed no false positive result. Conclusion The current study highlights the potential of LAMP test for the specific diagnosis of tubercular lymphadenitis in FNAC samples from HIV-infected patients, especially when cytology is either non-conclusive or non-available. Though MPCR had a higher sensitivity than LAMP assay, the added advantages of low cost, minimal technical expertise and simplicity of procedure make LAMP assay a suitable diagnostic test in resource-limited settings.

    更新日期:2019-11-04
  • A novel approach for detection of brucella using a real-time recombinase polymerase amplification assay
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-18
    Lide Qin, Wenlong Nan, Yong Wang, Yueyong Zhang, Pengfei Tan, Yuqi Chen, Kairong Mao, Yiping Chen

    Brucella, the etiological agent of brucellosis, is an important zoonosis pathogen worldwide. Brucella infects humans and various domestic and wild animals, and represents a great threat to public health and animal husbandry. In the present study, we developed a real-time recombinase polymerase amplification (RPA) assay for the detection of Brucella. The assay targeted the bcsp31 gene of Brucella, and an RPA exo probe and a pair of primers were selected for assay validation. RPA sensitivity and specificity were evaluated using plasmid standards, Brucella representative strains, and non-Brucella strains. The RPA assay achieved a detection limit of 17 molecules in 95% of cases based on probit analysis, and could successfully distinguish 18 representative Brucella strains (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae and B. ovis), and four Brucella vaccine strains (A19, S19, S2 and M5). A total of 52 Brucella field strains were detected by real-time PCR and RPA in parallel, and compared with real-time PCR, the sensitivity of the RPA assay was 94% (49/52). Thus, this RPA assay may be a rapid, sensitive, and specific tool for the prevention and control of Brucellosis.

    更新日期:2019-11-04
  • GSTO1 regards as a meritorious regulator in cutaneous malignant melanoma cells
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-13
    Li-Kun Wang, Hai-Long Yue, Xiao-Jing Peng, Shu-Juan Zhang

    Background Glutathione S-transferase omega 1 (GSTO1), as a member of the glutathione S-transferase (GST) family genes, has been discovered to be up-regulated in several cancer cell lines which exhibited strong aggressiveness. However, the function of GSTO1 on cutaneous malignant melanoma (CMM) has not been illuminated. Methods Outcome of expression level and prognosis of GSTO1 were obtained from Oncomine and TCGA database. The specific effects of GSTO1 on the characteristics and regulatory mechanism of CMM cells were demonstrated by cell counting kit-8, colony formation, flow cytometry, and transwell assays in vitro. Western blot was employed to analyze the expression of proliferating cell nuclear antigen (PCNA), p53 and epithelial-to-mesenchymal (EMT) related proteins. Results We observed that GSTO1 was up-regulated in CMM samples when compared with the corresponding controls. Moreover, patients in CMM with high expression of GSTO1 were more likely to have a poor prognosis. Through in vitro experiments, silenced GSTO1 resulted in inhibition of CMM cells growth and aggressiveness, increased cell apoptosis, and blocked cell cycle. Finally, the expression of PCNA, p53 and EMT-related proteins were changed due to reduction of GSTO1. Conclusions To sum up, our outcomes exhibited that weakening GSTO1 reduced the proliferation and mobility of CMM cells, increased the apoptosis ability of CMM cells, and arrested cell cycle at G1 phase, which can be achieved by affecting the expression of PCNA, p53 and the EMT process. This discovery provided a new perspective for elucidating the mechanism of CMM, and offered theoretical support for searching clinical therapeutic targets in the future.

    更新日期:2019-11-04
  • Development and evaluation of a direct TaqMan qPCR assay for the rapid detection of diverse carnivore amdoparvoviruses
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-12
    Yan-Hong Wu, Tao Wei, Xiu-Ting Zhang, Yong-Qiang Zhao, Jian-Ke Wang, Li Cong, Bao-Zeng Xu, Xi-Qun Shao

    Amdoparvoviruses infect carnivore species, including mink, raccoon dog, fox, skunk, and red panda. Amdoparvovirus infection is a major cause of morbidity and mortality in farmed minks. Here, we developed a direct TaqMan qPCR assay for detection and quantification of carnivore amdoparvoviruses by using three primers and one probe based on the conserved VP2 gene. The detection limit for Aleutian mink disease virus (AMDV) and Raccoon dog and arctic fox amdoparvovirus (RFAV) were 4.06 × 101 copies/μl and 2.93 × 101 copies/μl, respectively. Both intra- and inter-assay variability were less than 2%. Among 74 carnivore samples, the positive rates for amdoparvoviruses were 62.2% (46/74) by direct TaqMan qPCR, while only 40.5% (30/74) by SYBR Green I qPCR. This result suggests that the direct TaqMan qPCR was more sensitive than the SYBR Green I qPCR. Additionally, the direct TaqMan qPCR is a rapid and sensitive method for liquid samples at microliter level as the assay employed the direct alkaline lysis method to obtain viral DNA and, therefore, eliminated the cumbersome steps in extracting DNA. Overall, the direct TaqMan qPCR assay possessed high specificity, sensitivity, and reproducibility, indicating that it can be used as a powerful tool for detection and quantification of various carnivore amdoparvoviruses in epidemiological and pathogenesis studies.

    更新日期:2019-11-04
  • Designing self-assembled peptide nanovaccine against Streptococcus pneumoniae: An in silico strategy
    Mol. Cell. Probes (IF 2.511) Pub Date : 2019-09-11
    Hesam Dorosti, Mahboobeh Eslami, Navid Nezafat, Fardin Fadaei, Younes Ghasemi

    Streptococcus pneumoniae is the main cause of diseases such as meningitis, pneumoniae and sepsis, especially in children and old people. Due to costly antibiotic treatment, and increasing resistance of pneumococcus, developing high-efficient protective vaccine against this pathogen is an urgent need. Although the pneumoniae polysaccharide vaccine (PPV) and pneumonia conjugate vaccines (PCV) are the efficient pneumococcal vaccine in children and adult groups, but the serotype replacement of S. pneumoniae strains causes the reduction in efficacy of such vaccines. For overcoming the aforesaid drawbacks epitope-based vaccines are introduced as the relevant alternative. In our previous research, the epitope vaccine was designed based on immunodominant epitopes from PspA, CbpA antigens as cellular stimulants and PhtD, PiuA as humoral stimulants. Because the low immunogenicity is the main disadvantage of epitope vaccine, in the current study, we applied coiled-coil self-assembled structures for developing our vaccine. Recently, self-assembled peptide nanoparticles (SAPNs) have gained much attention in the field of vaccine development due to their multivalency, self-adjuvanticity, biocompatibility, and size similarity to pathogen. In this regard, the final designed vaccine is comprised of cytotoxic T lymphocytes (CTL) epitopes from PspA and CbpA, helper T lymphocytes (HTL) epitopes from PhtD and PiuA, the pentamer and trimmer oligomeric domains form 5-stranded and 3-stranded coiled-coils as self-assembled scaffold, Diphtheria toxoids (DTD) as a universal T-helper, which fused to each other with appropriate linkers. The four different arrangements based on the order of above-mentioned compartments were constructed, and each of them were modeled, and validated to find the 3D structure. The structural, physicochemical, and immunoinformatics analyses of final vaccine construct represented that our vaccine could stimulate potent immune response against S. pneumoniae; however, the potency of that should be approved via various in vivo and in vitro immunological tests.

    更新日期:2019-11-04
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