Real-time Polymerase Chain Reaction Quantification Research Articles

Screening and Validation of Key Genes of Autophagy in Acute Myocardial Infarction Based on Bioinformatics.

Autophagy plays a significant role in the development of acute myocardial infarction (AMI), and cardiomyocyte autophagy is of major importance in maintaining cardiac function. We aimed to identify key genes associated with autophagy in AMI through bioinformatics analysis and verify them through clinical validation. We downloaded an AMI expression profile dataset GSE166780 from Gene Expression Omnibus (GEO). Autophagy-associated genes potentially differentially expressed in AMI were screened using R software. Then, to identify key autophagy-related genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, protein-protein interaction (PPI) analysis, Receiver Operating Characteristic (ROC) curve analysis, and correlation analysis were performed on the differentially expressed autophagy-related genes in AMI. Finally, we used quantificational real-time polymerase chain reaction (qRT-PCR) to verify the RNA expression of the screened key genes. TSC2, HSPA8, and HIF1A were screened out as key autophagy-related genes. qRT-PCR results showed that the expression levels of HSPA8 and TSC2 in AMI blood samples were lower, while the expression level of HIF1A was higher than that in the healthy controls. TSC2, HSPA8, and HIF1A were identified as key autophagy-related genes in this study. They may influence the development of AMI through autophagy. These findings may help deepen our understanding of AMI and may be useful for the treatment of AMI.

Testosterone promotes the migration, invasion and EMT process of papillary thyroid carcinoma by up-regulating Tnnt1

PurposeTo explore the key genes and molecular pathways in the progression of thyroid papillary carcinoma (PTC) promoted by testosterone using RNA-sequencing technology, and to provide new drug targets for improving the therapeutic effect of PTC.MethodsOrchiectomy (ORX) was carried out to construct ORX mouse models. TPC-1 cells were subcutaneously injected for PTC formation in mice, and the tumor tissues were collected for RNA-seq. The key genes were screened by bioinformatics technology. Tnnt1 expression in PTC cells was knocked down or overexpressed by transfection. Cell counting kit-8 (CCK-8), colony formation assay, scratch assay and transwell assay were adopted, respectively, for the detection of cell proliferation, colony formation, migration and invasion. Besides, quantification real-time polymerase chain reaction (qRT-PCR) and western blot were utilized to determine the mRNA and protein expression levels of genes in tissues or cells.ResultsBoth estradiol and testosterone promoted the growth of PTC xenografts. The key gene Tnnt1 was screened and obtained by bioinformatics technology. Functional analysis revealed that overexpression of Tnnt1 could markedly promote the proliferation, colony formation, migration, invasion, and epithelial-to-mesenchymal transition (EMT) process of PTC cells, as well as could activate p38/JNK pathway. In addition, si-Tnt1 was able to inhibit the cancer-promoting effect of testosterone.ConclusionBased on the outcomes of bioinformatics and basic experiments, it is found that testosterone can promote malignant behaviors such as growth, migration, invasion and EMT process of PTC by up-regulating Tnnt1 expression. In addition, the function of testosterone may be achieved by activating p38/JNK signaling pathway.

DDX43 mRNA expression and protein levels in relation to clinicopathological profile of breast cancer.

Breast cancer (BC) is the most often diagnosed cancer in women globally. Cancer cells appear to rely heavily on RNA helicases. DDX43 is one of DEAD- box RNA helicase family members. But, the relationship between clinicopathological, prognostic significance in different BC subtypes and DDX43 expression remains unclear. Therefore, the purpose of this study was to assess the clinicopathological significance of DDX43 protein and mRNA expression in different BC subtypes. A total of 80 females newly diagnosed with BC and 20 control females that were age-matched were recruited for this study. DDX43 protein levels were measured by ELISA technique. We used a real-time polymerase chain reaction quantification (real-time PCR) to measure the levels of DDX43 mRNA expression. Levels of DDX43 protein and mRNA expression within BC patients had been compared to those of control subjects and correlated with clinicopathological data. The mean normalized serum levels of DDX43 protein were slightly higher in control than in both benign and malignant groups, but this result was non-significant. The mean normalized level of DDX43 mRNA expression was higher in the control than in both benign and malignant cases, although the results were not statistically significant and marginally significant, respectively. Moreover, the mean normalized level of DDX43 mRNA expression was significantly higher in benign than in malignant cases. In malignant cases, low DDX43 protein expression was linked to higher nuclear grade and invasive duct carcinoma (IDC), whereas high mRNA expression was linked to the aggressive types of breast cancer such as TNBC, higher tumor and nuclear grades. This study explored the potential of using blood DDX43 mRNA expression or protein levels, or both in clinical settings as a marker of disease progression in human breast cancer. DDX43 mRNA expression proposes a less invasive method for discriminating benign from malignant BC.

A comprehensive diagnostic scheme of morphological combined molecular methylation under bronchoscopy.

Methylated SHOX2 and RASSF1A genes are potential biomarkers for lung cancer diagnosis. Therefore, we explored the role of methylation detection combined with morphological bronchoscopic evaluation for lung cancer diagnosis. Bronchoscopy, methylation outcome, and pathological data were collected from 585 patients with lung cancer and 101 controls. The methylation status of the SHOX2 and RASSF1A genes were detected using real-time polymerase chain reaction quantification. Further, the sensitivity and area under the receiver operating characteristic curve of the three methods were analyzed. Among 686 patients, 57.1% had new lesions detected through bronchoscopy and 93.1% of these patients were diagnosed with malignant tumors. Besides, 42.9% of patients had no visible changes under bronchoscopy but there were still 74.8% of them diagnosed with malignant tumors. Bronchoscopy revealed that lung adenocarcinoma, lung squamous cell carcinoma, and small cell lung cancer mainly occurred in the upper and middle lobes. The sensitivity and specificity of methylation detection were 72.8% and 87.1% (vs. cytology 10.4% & 100%), respectively. Therefore, methylated SHOX2 and RASSF1A genes may be promising tumor markers in lung cancer diagnosis. Methylation detection can be an excellent supplementary tool for cytological diagnosis and, combined with bronchoscopy, could form a more effective diagnostic process.

Osteoinductive and Osteogenic Capacity of Freeze-Dried Bovine Bone Compared to Deproteinized Bovine Bone Mineral Scaffold in Human Umbilical Cord Mesenchymal Stem Cell Culture: An In Vitro Study.

Freeze-dried bovine bone scaffold (FDBB) or decellularized FDBB (dc-FDBB) was developed as an ideal scaffold with osteoinductive properties. This research aims to compare the osteoinductive properties marked by the expression of runt-related transcription factor-2 (RUNX2) and Osterix (OSX) and the osteogenic capacity of these scaffolds imbued with human umbilical cord mesenchymal stem cells (hUCMSCs). This study was performed in five experimental groups: a negative control group (C-) of hUCMSCs with a normal growth medium, a positive control group (C + ) of hUCMSCs with an osteogenic medium, experimental group 1 (E1) with an FDBB conditioned medium (CM), and experimental group 2 (E2) with a dc-FDBB-CM, and a third experimental group (E3) consisting of a DBBM-CM. Alizarin red staining was performed to qualitatively assess osteoinductive capacity. RUNX2 and OSX expression was quantified using real-time quantification polymerase chain reaction with two replications on day six (D6) and day 12 (D12) as fold changes. This experiment revealed that hUCMSCs were positively expressed by CD73, CD90, and CD105 but were not expressed by CD34. Alizarin red staining showed that E1 had the most calcium deposition on D6 and D12, followed by E3 and then E2 The RUNX2 and OSX expression was higher in E1 but this difference was not significant. The OSX expression in E1,E2,E3 was lower on D12 and C+ of OSX had the highest expression. There was a significant difference of fold change measured between all groups (p < 0.05), and there was no significant difference between any of the groups treated with OSX and RUNX2 on D6 and D12. FDBB osteoinduction and osteogenic capacity were higher when compared with DBBM and dc-FDBB.

Krüppel-like Factor 6 Suppresses the Progression of Pancreatic Cancer by Upregulating Activating Transcription Factor 3

As a member of the Krüppel-like factor (KLFs) family, Krüppel-like factor 6 (KLF6) plays a critical role in regulating key cellular functions. Presently, scholars have proved the important role of KLF6 in the tumorigenesis of certain cancers through a large number of experiments. However, gaps still remain in our knowledge of the role of KLF6 in pancreatic cancer (PAAD). Therefore, this paper mainly investigates the role of KLF6 in the progression of pancreatic cancer. The expression pattern of KLF6 in pancreatic cancer was explored in pancreatic cancer tissues and cell lines. Then, we investigated the prognostic value of KLF6 in pancreatic cancer by immunohistochemical assays. Next, Cell Counting Kit-8 (CCK8) and clone information assays were employed to explore the proliferation of PAAD affected by KLF6. The metastasis and epithelial-mesenchymal transition (EMT) abilities affected by KLF6 were identified through transwell invasion as well as migration assays and western blots. Finally, the TRRUST tool was used to analyze the potential targeted genes of KLF6. The results were verified by Quantificational Real-time Polymerase Chain Reaction (qRT-PCR), western blot and rescue assays. KLF6 expresses lowly in pancreatic cancer compared to corresponding normal tissues and relates to poor survival times. Overexpression of KLF6 inhibits the proliferation, metastasis, and EMT progression in pancreatic cancer cells. Further studies suggest that KLF6 could upregulate ATF3 in PAAD. Our results suggest that KLF6 can be a useful factor in predicting the prognosis of PAAD patients and that it inhibits the progression of pancreatic cancer by upregulating activating transcription factor 3 (ATF3).

Effects of DNA extraction methods on the real time PCR quantification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in chicken feces and ceca contents

Polymerase chain reaction (PCR) method was coupled with a DNA extraction to enumerate Campylobacter spp. from poultry gastrointestinal tract samples. Three experiments were conducted that included: 1) Development of a DNA standard curve related to bacterial DNA primers; 2) Design of a cell/genomic DNA extraction protocol to isolate Campylobacter spp. DNA from complex samples such as poultry feces; and 3) Comparison of PCR quantification to standard plate count methodology. The standard curve using primers for Campylobacter spp. was created for DNA extracted from environmental isolates with a linear range (R2 > 0.95) and with a high specificity for C. coli and C. jejuni recovered from poultry, swine and laboratory isolates. A 2-step extraction process of bacterial DNA from poultry feces was developed in which the cells were first concentrated using a gradient-centrifugation step followed by comparison of 4 DNA extraction methods. Two commercial DNA extraction methods (Zymo Research Quick DNA, and Invitrogen magnetic separation), a traditional phenol-chloroform DNA extraction method using proteinase K to inactivate DNAses, and an in-house isolation method for DNA extraction based on chaotropic salts were used. The middle gradient layer recovered 89% to 98% of the bacteria cells from the sample, with recovery dependent upon the Campylobacter genus. The 4 DNA extractions methods recovered 112 to 302 ug/nL of DNA. Finally, the qPCR and standard plate methods were highly correlated for enumerating Campylobacter spp. in the 2.0 to 8.0-log CFU range. Analyses of the results from this study demonstrate that the combination of the standard curve for Campylobacter spp. DNA primers, the gradient cell concentration method and DNA extraction techniques with qPCR can be used to enumerate Campylobacter spp. from poultry samples with findings similar those of traditional plate count methodology.

Circular RNA circ_0114876 regulates osteoarthritis through upregulating ADAM10 via targeting miR-1227-3p

BackgroundOsteoarthritis (OA) was a chronic degenerative joint disease. The dysregulation of circular RNAs (circRNAs) has been identified in OA progression. However, the function and regulation mechanism of circ_0114876 in OA remains largely unknown. MethodFirstly, we used LPS-treated C28/I2 cells as a cellular model of OA. Quantificational real-time polymerase chain reaction (qRT-PCR) was used to determine the expression levels of circ_0114876, miRNA-1227-3p, and ADAM10 in OA chondrocytes. Cell Counting Kit-8 (CCK8), 5-ethynyl-20-deoxyuridine (EdU) incorporation assays, flow cytometry, Enzyme-linked immunosorbent assay (ELISA) kit, and western blot were applied to confirm cell proliferation, apoptosis, inflammation, and extracellular matrix.of circ_0114876 in vitro. The interaction between circ_0114876 and its downstream target (miR-1227-3p) and mRNA target ADAM metallopeptidase domain 10 (ADAM10), was evaluated by luciferase assay and RNA immunoprecipitation (RIP) assay. ResultCirc_0114876 and ADAM10 were upregulated and miR-1227-3p was decreased in OA tissues and LPS-treated chondrocytes. Low expression of circ_0114876 promoted proliferation and inhibited apoptosis, inflammation, and extracellular matrix of the LPS-treated chondrocytes. Mechanistically, circ_0114876 functioned in human chondrocytes through targeting miR-1227-3p and ADAM10. Furthermore, miRNA-1227-3p inhibitor reversed the effect of circ_0114876 knockdown on the OA chondrocytes, and ADAM10 overexpression reversed the effect of miR-1227-3p mimic on the OA chondrocytes. ConclusionCirc_0114876 was increased in OA tissues and cells. Circ_0114876 facilitated the progression in the LPS-induced OA cell model via regulating the miR-1227-3p/ADAM10 axis. This study would provide a potentially effective therapeutic strategy for OA progression.

Notch signaling-induced cyclin d1 in diabetes ameliorating effects of the isolated polysaccharide from Rosa canina: In vitro and in vivo studies.

Notch signaling has a role in the expansion of the pancreas and the pathogenesis of diabetes. Modulation of Notch signaling by natural products seems to pave the way for treating diabetes. This research aimed to scrutinize the involvement of the Notch cascade in the diabetes-ameliorating effects of an isolated polysaccharide from Rosa canina. The isolated polysaccharide was characterized using Fourier transform infrared, nuclear magnetic resonance, high-performance gel-permeation chromatography, and liquid chromatography with tandem mass spectrometry techniques. Rat pancreatic β cells and STZ-induced diabetic rats were treated with the isolated polysaccharide. MTT assay, cell cycle analysis, quantative realtime-polymerase chain reaction, immunohistochemistry, and immunoblotting were used to reveal the growth and the expression levels of Notch1, DLL4, Jagged-1, hes1, Ins-1, Pdx-1, and cyclin d1 in treated and untreated pancreatic cells and tissues. The ameliorating effect of the polysaccharide in STZ-treated cells was accomplished by upregulation of cyclin d1 and hes1 as well as cell cycle progression. Notch inhibition by LY-411575 was associated with the downregulation of cyclin d1 which upregulates with polysaccharide treatment. The significant expression of cyclin d1 (90%) and nuclear expression of hes1 in the pancreas of the polysaccharide group were accompanied by improvement of hyperglycemia and associated biochemical factors as well as regeneration of islet cells as compared to untreated diabetic rats. Based on these findings, upregulation of Notch signaling-induced cyclin d1 could be proposed as the underlying diabetes-reducing effects of the isolated polysaccharide derivative implying that cyclin d1 actuation through activation of the Notch-DLL4 circuit may play the causal role in the treatment of diabetes.

Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis.

To investigate the therapeutic effect of gentisic acid (GA) on rheumatoid arthritis (RA) based on the miR-19b-3p/RAF1 axis. The cell counting kit-8 method was used to detect the growth inhibitory effect of different concentrations of GA on MH7A cells, and the drug concentration of GA was determined in the experiment. The quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-19b-3p and RAF1. RAF1, extracellular regulated protein kinases1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were examined by Western blotting. Three methods (dual-luciferase assay, qRT-PCR and Western blot analysis) were used to verify miR-19b-3p targeting RAF1. Flow cytometry was performed to detect MH7A cell apoptosis. Transwell and wound healing assays were used to determine the invasion and migration capacities of MH7A cells. The growth of MH7A cells was gradually inhibited with increasing GA concentration. When the GA concentration exceeded 80 mmol/L, GA was significantly cytotoxic to MH7A cells, so the half maximal inhibitory concentration of GA for MH7A cells was calculated as 67.019 mmol/L. GA upregulated miR-19b-3p expression, downregulated RAF1 expression, inhibited ERK1/2 phosphorylation, induced MH7A cell apoptosis and suppressed MH7A cell invasion and migration (P<0.05 or P<0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (P<0.05 or P<0.01). The miR-19b-3p inhibitor upregulated RAF1 expression and ERK1/2 phosphorylation, suppressed MH7A cell apoptosis and induced MH7A cell invasion and migration (P<0.01). GA regulated miR-19b-3p/RAF1 axis to mediate ERK pathway and inhibit the development of RA.

Genome-wide identification and characterization of the JAZ gene family and its expression patterns under various abiotic stresses in Sorghum bicolor

The jasmonate ZIM domain (JAZ) protein belongs to the TIFY ((TIF[F/Y]XG) domain protein) family, which is composed of several plant-specific proteins that play important roles in plant growth, development, and defense responses. However, the mechanism of the sorghum JAZ family in response to abiotic stress remains unclear. In the present study, a total of 17 JAZ genes were identified in sorghum using a Hidden Markov Model search. In addition, real-time quantification polymerase chain reaction (RT-qPCR) was used to analyze the gene expression patterns under abiotic stress. Based on phylogenetic tree analysis, the sorghum JAZ proteins were mainly divided into nine subfamilies. A promoter analysis revealed that the SbJAZ family contains diverse types of promoter cis-acting elements, indicating that JAZ proteins function in multiple pathways upon stress stimulation in plants. According to RT-qPCR, SbJAZ gene expression is tissue-specific. Additionally, under cold, hot, polyethylene glycol, jasmonic acid, abscisic acid, and gibberellin treatments, the expression patterns of SbJAZ genes were distinctly different, indicating that the expression of SbJAZ genes may be coordinated with different stresses. Furthermore, the overexpression of SbJAZ1 in Escherichia coli was found to promote the growth of recombinant cells under abiotic stresses, such as PEG 6000, NaCl, and 40°C treatments. Altogether, our findings help us to better understand the potential molecular mechanisms of the SbJAZ family in sorghum in response to abiotic stresses.

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states

Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be ‘aroused’ to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.

Comparative real-time PCR quantification of cytomegalovirus in severe early childhood caries and caries-free children

The present study was undertaken to evaluate cytomegalovirus (CMV) load in carious lesions in children with severe early childhood caries (S-ECC) and compare it with caries-free (CF), healthy children, by quantitative real-time polymerase chain reaction (PCR). Dental plaque samples were collected from 57 subjects including S-ECC (n = 33) and CF (n = 24) patients. The two groups were matched in terms of age, gender, and socioeconomic status. After deoxyribonucleic acid (DNA) extraction, SYBR Green real-time PCR was used to quantify CMV DNA. The CMV DNA was detected in seven patients in theS-ECC group. The mean of decayed, missing, and filled tooth surfaces (dmfs) was 11.90 ± 5.95 in the S-ECC group. There was no significant difference in gender between the two groups. There was a significant relationship between CMV positivity in children and theS-ECC group (p = 0.043). Results of this study suggest a relationship between the presence of CMV in the oral cavity and S-ECC. We can design clinical trials to confirm these results and improve children's dental care.

Construction and Validation of a 6-Ferroptosis Related Gene Signature for Prognosis and Immune Landscape Prediction in Melanoma.

Ferroptosis is a newly discovered form of non-apoptotic cell death that relies on iron-mediated oxidative damage, playing a crucial role in the progression and therapy resistance of melanoma. Hence, the potential value of ferroptosis-related genes (FRGs) as a prognostic model and therapeutic target in melanoma requires further investigation. In this study, the relationship between FRGs and melanoma was revealed by analyzing the mRNA expression profiles from The Cancer Genome Atlas (TCGA) and Gene Expression Synthesis (GEO). A 6-FRGs signature was constructed by Univariate, multivariate, and lasso Cox regression analyses in the TCGA cohort. The GEO database was used to validate the efficacy of the signature. The protein and mRNA expression level of the signature genes were examined in real-world melanoma tissues via immunohistochemical and quantificational real-time polymerase chain reaction (qRT-PCR). Functional enrichment analysis and immune-related analysis were conducted to identify the potential biological functions and pathways of the signature. Ten putative small molecule drugs were predicted by Connectivity Map (CMAP). As a result, a 6-FRGs signature was constructed to stratify melanoma patients into two risk groups. Compared with the low-risk group, patients in the high-risk group had a worse prognosis and a lower ImmuneScore. Immune-related pathways were enriched in the low-risk group. Immune Function and immune cell infiltration of the low-risk group were significantly higher than that of the high-risk group. The differential expression of these six FRGs in melanoma and adjacent normal tissues was confirmed. Moreover, higher expression of immune checkpoint molecules and a greater sensitivity to immunotherapy were observed in the low-risk group. Some small molecular drugs in the CMAP database hold the potential to treat melanoma. Overall, we identified a novel FRGs signature for prognostic prediction in melanoma. Based on the signature-related immune infiltration landscape found in our study, targeting the FRGs might be a therapeutic alternative for melanoma.

A Crucial Angiogenesis-Associated Gene MEOX2 Could Be a Promising Biomarker Candidate for Breast Cancer.

BackgroundAngiogenesis plays a critical role in the growth and metastasis of breast cancer and angiogenesis inhibition has become an effective strategy for cancer therapy. Our study aimed to clarify the key candidate genes and pathways related to breast cancer angiogenesis.MethodsDifferentially expressed genes (DEGs) in the raw breast cancer (BRCA) gene dataset from the Cancer Genome Atlas (TCGA) database were identified and gene ontology analysis of the DEGs was performed. Hub genes were subsequently determined using the Gene Expression Omnibus database. The expression of the mesenchyme homeobox 2 (MEOX2) in breast cancer cells and tissues was assessed by quantification real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. The prognostic value of the MEOX2 gene in breast cancer tissue was evaluated with the Kaplan-Meier plotter.ResultsA total of 61 angiogenesis-related DEGs were identified in the TCGA dataset, among which the gene MEOX2 was significantly down-regulated. GO functional annotation and pathway enrichment analyses showed that MEOX2 was significantly enriched in the regulation of vasculature development. The IHC results confirmed that MEOX2 expression was repressed in breast cancer tissues and the relatively low level indicated the tissue was densely vascularized. Moreover, MEOX2 expression was significantly elevated in breast cancer cells after treatment with cisplatin (DDP) and epirubicin (EPI). Finally, the Kaplan-Meier plotter confirmed that higher expression levels of MEOX2 were related to better overall survival.ConclusionOur study revealed that the angiogenesis-associated gene MEOX2 can be used as a novel biomarker for breast cancer diagnosis and clinical therapy.

Bone marrow mesenchymal stem cells attenuate pyroptosis lipopolysaccharide-induced renal injury rats

To investigate the effect and mechanism of bone marrow mesenchymal stem cell (BMSC) on pyroptosis of rats with kidney injury. Bone marrow of 4-5 week-old female Sprague-Dawley (SD) rats was isolated in vitro and BMSC was obtained. The third generations of BMSC were used to further experiments. Fifteen 6 week-old SD rats were cluster-randomized divided into control group, kidney injury group and BMSC group (5 rats in each group). Rats in kidney injury group were injected with lipopolysaccharide (LPS) 1 mg/kg via tail vein; the control group was given the same amount of normal saline. BMSC group was injected with 0.5 mL BMSC (including 2×106 BMSC) via tail vein after modeling; the kidney injury group received the same amount of normal saline. On day 3 after these injections, serum creatinine (SCr) was detected by picric acid method, and blood urea nitrogen (BUN) was detected by diacetyl monoxime. The levels of cystatin C (Cys C), interleukins (IL-1β and IL-18) in blood were detected by enzyme-linked immunosorbent assay (ELISA). The rats were then sacrificed and their kidneys were removed for subsequent detection. The mRNA expression levels of NOD-like receptor protein 3 (NLRP3) and cysteinyl aspartate-specific protease-1 (caspase-1) of kidney were detected by quantificational real-time quantitative polymerase chain reaction (qRT-PCR). The protein expression levels of NLRP3 and caspase-1 of kidney were detected by Western blotting. In vitro, after bone marrow cell suspension was cultured for 24 hours, a large number of round adherent cells and suspended cells appeared in each culture flask. After 4-5 days of culture, a large number of long spindle cells adhered to the wall, and there were still obvious impurity cells. After trypsin digestion and passage to the third generation, the long spindle adherent cells grew mainly in the culture flask and were basically purified as BMSC. In vivo, compared with the control group, the levels of SCr, BUN, Cys C, IL-1β and IL-18 in kidney injury group were increased [SCr (μmol/L): 85.22±2.29 vs. 21.80±0.59, BUN (mmol/L): 11.50±0.64 vs. 5.86±0.83, Cys C (mg/L): 0.13±0.01 vs. 0.11±0.02, IL-1β (ng/L): 31.49±1.42 vs. 4.74±0.49, IL-18 (ng/L): 29.01±1.95 vs. 1.52±0.03, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly increased [NLRP3 mRNA (2-ΔΔCt): 3.635±0.296 vs. 1.000±0.002, caspase-1 mRNA (2-ΔΔCt): 4.020±0.228 vs. 1.001±0.003; NLRP3 protein (NLRP3/β-actin): 1.560±0.868 vs. 0.902±0.036, caspase-1 protein (caspase-1/β-actin): 1.392±0.097 vs. 0.895±0.046, all P < 0.05]. Compared with kidney injury group, the levels of SCr, BUN, IL-1β and IL-18 in BMSC group were significantly decreased [SCr (μmol/L): 51.64±3.84 vs. 85.22±2.29, BUN (mmol/L): 9.90±0.46 vs. 11.50±0.64, IL-1β (ng/L): 24.20±1.45 vs. 31.49±1.42, IL-18 (ng/L): 12.97±1.25 vs. 29.01±1.95, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly decreased [NLRP3 mRNA (2-ΔΔCt): 1.488±0.136 vs. 3.635±0.296, caspase-1 mRNA (2-ΔΔCt): 1.643±0.143 vs. 4.020±0.228; NLRP3 protein (NLRP3/β-actin): 1.227±0.053 vs. 1.560±0.868, caspase-1 protein (caspase-1/β-actin): 1.159±0.107 vs. 1.392±0.097, all P < 0.05]. In vivo, BMSC may attenuate pyroptosis in LPS-induced kidney injury rats.

Two bicistronic DNA vaccines against Vibrio anguillarum and the immune effects on flounder Paralichthys olivaceus.

Chemokines are cytokines that can promote the activation and migration of immune cells, and increase the recognition of antigen by antigen-presenting cells (APC). Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of flounder after immunization. To explore the improvement of chemokines on the efficiency of OmpK vaccine, two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the flounder were investigated by reverse transcription polymerase chain reaction (RT-PCR), indirect immunofluorescent assay (IFA), H&E staining, flow cytometry (FCM), and quantificational real-time polymerase chain reaction (qRT-PCR). pBudCE4.1 plasmid as an expression vector, bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene (p-OmpK-CCL4), or Ompk gene and CC-motif ligand 19 gene (p-OmpK-CCL19) were successfully constructed. The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/CCL19 proteins of flounder both in vitro and in vivo. After immunization, a large number of leucocytes in muscle were recruited at the injection site in treatment groups. The constructed vaccines induced significant increases in CD4-1+ and CD4-2+ T lymphocytes, and sIgM+ B lymphocytes in peripheral blood, spleen, and head kidney. The percentage of T lymphocytes peaked on the 14th post-vaccination day whereas that of B lymphocytes peaked in the 6th post-vaccination week. Moreover, the expression profiles of 10 immune-related genes increased in muscles around the injection site, spleen, and head kidney. After the challenge, p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival (RPS) of 74.1% and 63.3%, respectively, higher than p-OmpK alone (40.8%). In conclusion, both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity. CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.Electronic supplementary materialSupplementary material (Supplementary Material) is available in the online version of this article at 10.1007/s00343-021-1092-z.

Transcriptome-wide Identification and Characterization of microRNAs and Their Targets in a Highly Adaptable Conifer Platycladus orientalis

MicroRNAs (miRNAs) are short noncoding RNAs (20–25 nucleotides) that regulate gene expression posttranscriptionally. However, identification and characterization of miRNAs remain limited for conifer species. In this study, we applied transcriptome-wide miRNAs sequencing to a conifer species Platycladus orientalis, which is highly adaptable to a wide range of environmental adversities, including drought, barren soil, and mild salinity. A total of 17,181,542 raw reads were obtained from the Illumina sequencing platform; 31 conserved and 91 novel miRNAs were identified, and their unique characteristics were further analyzed. Ten randomly selected miRNAs were validated by quantificational real-time polymerase chain reaction. Through miRNA target predictions based on psRNATarget, 2331 unique mRNAs were predicted to be targets of P. orientalis miRNAs that involved in 187 metabolic pathways in KEGG database. These targets included not only important transcription factors (e.g., class III homeodomain leucine zipper targeted by por-miR166d) but also indispensable nontranscriptional factor proteins (i.e., por-miR482a-3p regulated nucleotide-binding site leucine-rich repeat protein). Interestingly, six miRNAs (por-miR16, -miR44, -miR60-5p, -miR69–3p, -miR166b-5p, and -miR395c) were found in adaptation-related pathways (e.g., drought), indicating their possible involved in this species’ stress-tolerance characteristics. The present study provided essential information for understanding the regulatory role of miRNAs in P. orientalis and sheds light on their possible use in tree improvement for stress tolerance.

Silencing of GhKEA4 and GhKEA12 Revealed Their Potential Functions Under Salt and Potassium Stresses in Upland Cotton.

The K+ efflux antiporter (KEA) mediates intracellular K+ and H+ homeostasis to improve salt tolerance in plants. However, the knowledge of KEA gene family in cotton is largely absent. In the present study, 8, 8, 15, and 16 putative KEA genes were identified in Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These KEA genes were classified into three subfamilies, and members from the same subfamilies showed similar motif compositions and gene structure characteristics. Some hormone response elements and stress response elements were identified in the upstream 2000 bp sequence of GhKEAs. Transcriptome data showed that most of the GhKEAs were highly expressed in roots and stems. The quantificational real-time polymerase chain reaction (qRT-PCR) results showed that most of the GhKEAs responded to low potassium, salt and drought stresses. Virus-induced gene silencing (VIGS) experiments demonstrated that under salt stress, after silencing genes GhKEA4 and GhKEA12, the chlorophyll content, proline content, soluble sugar content, peroxidase (POD) activity and catalase (CAT) activity were significantly decreased, and the Na+/K+ ratio was extremely significantly increased in leaves, leading to greater salt sensitivity. Under high potassium stress, cotton plants silenced for the GhKEA4 could still maintain a more stable Na+ and K+ balance, and the activity of transporting potassium ions from roots into leaves was reduced silenced for GhKEA12. Under low potassium stress, silencing the GhKEA4 increased the activity of transporting potassium ions to shoots, and silencing the GhKEA12 increased the ability of absorbing potassium ions, but accumulated more Na+ in leaves. These results provided a basis for further studies on the biological roles of KEA genes in cotton development and adaptation to stress conditions.

Detection and Real-time PCR Assay for the Quantification of Carbapenemase Gene blablaNDM-1 in Hospital Effluent

This study aims to isolate gram-negative bacteria (GNB) harboring the gene NDM-1 from the tertiary care hospital effluents. Also, aims to evaluate the relative copy number of blaNDM-1 carried by the positive isolates. The study isolated 215 GNB from 40 effluent samples. The antibiotic susceptibility tests for carbapenems were performed using disc diffusion assay. The isolates resistant to either meropenem or imipenem were checked for the existence of MBL by phenotypic methods. The isolates carrying NDM-1 gene were genotypically confirmed by Polymerase chain reaction (PCR). The gene copy number of blaNDM- were determined by quantative real-time PCR. A total of 22 isolates showed phenotypic resistance to carbapenems and were characterized by biochemical methods. Among them, 12 harbored NDM-1 gene by PCR; these bacteria were subjected to qPCR for determining the absolute copy numbers of the NDM-1 gene on it. The gene abundance in the strains was in the range of 3.28× 105 to 6.05× 106 copies/ ng of DNA. Hospital effluents are important pool of antibiotic-resistant bacteria harboring the blaNDM-1 and infections caused by these bacteria are difficult to treat. Hence, the present study stresses the need for stringent antibiotic use and efficient wastewater treatment policies in these hospital settings, which is paramount in achieving sustainable health.