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This study investigated the effects of amygdalin (AMY, a cyanogenic glycoside widely distributed in the fruits and seeds of Rosaceae plants) on cardiac performance and ventricular remodeling in a rat model of myocardial infarction (MI). We also investigated whether the combination of AMY with exercise training (ExT) has a beneficial synergistic effect in treating MI rats. MI was induced by the ligation of the left anterior descending coronary artery in male SD rats. ExT or AMY treatment was started 1 week after MI and continued for 1 week (short-term) or 8 weeks (long-term). Cardiac function was evaluated by echocardiographic and hemodynamic parameters. Heart tissues were harvested and subjected to 2,3,5-triphenyl-tetrazolium chloride, Masson's trichrome, hematoxylin-eosin, and immunohistochemical staining. Gene expression was determined by quantitative polymerase chain reaction. Western blot gave a qualitative assessment of protein levels. AMY or ExT improved cardiac function and reduced infarct size in MI rats. AMY or ExT also suppressed myocardial fibrosis and attenuated inflammation in the infarct border zone of hearts from MI rats, as evidenced by inhibition of collagen deposition, inflammatory cell infiltration, and pro-inflammatory markers (interleukin 1β, interleukin 6, tumor necrosis factor-α, and cyclooxygenase 2). Notably, the effects of AMY combined with ExT were superior to those of AMY alone or ExT alone. Mechanistically, these beneficial functions were correlated with the inhibition of MI-induced activation of the transforming growth factor-β/Smad pathway. Collectively, AMY and ExT exert a synergistic effect on improving cardiac performance and ameliorating cardiac inflammation and fibrosis after MI, and the effects of long-term intervention were better than short-term intervention. [ABSTRACT FROM AUTHOR]

Objective: To evaluate toxicity of raw extract of Panax notoginseng (rPN) and decocted extract of PN (dPN) by a toxicological assay using zebrafish larvae, and explore the mechanism by RNA sequencing assay. Methods: Zebrafish larvae was used to evaluate acute toxicity of PN in two forms: rPN and dPN. Three doses (0.5, 1.5, and 5.0 µ g/mL) of dPN were used to treat zebrafishes for evaluating the developmental toxicity. Behavior abnormalities, body weight, body length and number of vertebral roots were used as specific phenotypic endpoints. RNA sequencing (RNA-seq) assay was applied to clarify the mechanism of acute toxicity, followed by real time PCR (qPCR) for verification. High performance liquid chromatography analysis was performed to determine the chemoprofile of this herb. Results: The acute toxicity result showed that rPN exerted higher acute toxicity than dPN in inducing death of larval zebrafishes (P<0.01). After daily oral intake for 21 days, dPN at doses of 0.5, 1.5 and 5.0 µ g/mL decreased the body weight, body length, and vertebral number of larval zebrafishes, indicating developmental toxicity of dPN. No other adverse outcome was observed during the experimental period. RNA-seq data revealed 38 genes differentially expressed in dPN-treated zebrafishes, of which carboxypeptidase A1 (cpa1) and opioid growth factor receptor-like 2 (ogfrl2) were identified as functional genes in regulating body development of zebrafishes. qPCR data showed that dPN significantly down-regulated the mRNA expressions of cpa1 and ogfrl2 (both P<0.01), verifying cpa1 and ogfrl2 as target genes for dPN. Conclusion: This report uncovers the developmental toxicity of dPN, suggesting potential risk of its clinical application in children. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) function as potential diagnostic biomarkers in various cancers. This study aimed to evaluate the roles of miR‐205‐5p in lung cancer progression and diagnosis. Materials and Methods: MiR‐205‐5p was detected by quantitative real‐time PCR. The effect of miR‐205‐5p on cell proliferation and metastasis was estimated by MTT and flow cytometry. The expression of TP53INP1 and related genes was analyzed by immunoblotting. The diagnostic value of miR‐205‐5p was analyzed using receiver operating characteristic (ROC) curve analysis, sensitivity, and specificity. Results: The miR‐205‐5p was increased in lung cancer tissues. MiR‐205‐5p mimics were promoted but its inhibitor suppressed cell proliferation and metastasis compared with control treatment in vitro and in vivo. By regulating the 3′ untranslated region, miR‐205‐5p could negatively regulate TP53INP1 expression, which further inhibited the expression of RB1 and P21, but increased that of cyclinD1. Moreover, the serum miR‐205‐5p levels of patients with lung cancer were significantly higher than those of normal controls, and they were correlated with patients' gender, drinking status, and clinical stage. The area under the ROC curve of serum miR‐205‐5p in the diagnosis of non‐small‐cell lung cancer was 0.8250, respectively. The finding supported its possession of high diagnostic efficiency for lung cancer. Conclusions: MiR‐205‐5p promoted lung cancer cell proliferation and metastasis by negatively regulating the novel target TP53INP1, which further affected the expression of P21, RB1, and cyclin D1. Serum miR‐205‐5p is a novel and valuable biomarker for lung cancer diagnosis. [ABSTRACT FROM AUTHOR]

We designed this study to assess if surgical safety can be improved by intraoperative use of intraocular lens (IOL) for cataract phacoemulsification. We performed phacoemulsification cataract removal on 401 patients. We randomly assigned these patients into three groups: the standard setting (Group I, n = 134), with reduced vacuum and flow rate (Group II, n = 137), and with IOL insertion before the last quadrant was emulsified with standard setting (Group III, n = 130). The primary outcomes included the risk of posterior capsular rupture (PCR), ultrasound time, energy, and complications. The secondary outcomes included central corneal thickness (CCT), CCT changes, endothelial cells (ETC) counting, ETC loss, and the best corrected distance visual acuity (BCVA) measured on day 1, day 7 and day 30. If PCR occurred, we emulsified the residual lens materials after insertion of IOL and clean of the prolapsed vitreous. We found that the risk of PCR in Group III (0/130) was lower than Group I (9/134, corrected relative risk (RR) = 18.44, 95% CI: 1.08–313.56) and Group II (3/137, corrected RR = 6.64, 95% CI: 0.35–27.41). Group III showed better BCVA on day 1 and 7, less ECC loss on day 7 and 30, and less CCT increase on day 1 and 7. No cases converted to extracapsular cataract extraction. No residual lens materials misdirected into vitreous cavity. Intraoperative use of IOL can improve surgical safety for dense cataract phacoemulsification. [ABSTRACT FROM AUTHOR]

Fine particulate matter (PM2.5) is an important environmental risk factor for cardiovascular diseases. However, little is known about the effects of PM2.5 on arteries. The present study investigated whether PM2.5 alters 5‐hydroxytryptamine (5‐HT) receptor expression and inflammatory mediators on rat mesenteric arteries, and examined the underlying mechanisms. Isolated rat mesenteric arteries segments were cultured with PM2.5 in the presence or absence of ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was monitored by a sensitive myograph. The expression of 5‐HT2A/1B receptors and inflammatory mediators were studied by a real‐time polymerase chain reaction and/or by immunohistochemistry. The phosphorylation of mitogen‐activated protein kinases (MAPK) pathway was detected by Western blot. Compared with the fresh or culture alone groups, 1.0 μg/mL PM2.5 cultured for 16 hours significantly enhanced contractile response induced by 5‐HT and increased 5‐HT2A receptor mRNA and protein expressions, indicating PM2.5 upregulates 5‐HT2A receptor. SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly decreased PM2.5‐induced elevated contraction and mRNA and protein expression of 5‐HT2A receptor. Cultured with PM2.5 significantly increased the mRNA expression of inflammatory mediators (NOS2, IL‐1β, and TNF‐α), while SB203580 decreased mRNA expression level of NOS2, IL‐1β, and TNF‐α. SP600125 (JNK inhibitor) decreased mRNA expression level of TNF‐α and IL‐1β. After PM2.5 exposure, the phosphorylation of p38 and ERK1/2 protein were increased. SB203580 and U0126 inhibited the PM2.5 caused increased phosphorylation protein of p38 and ERK1/2. In conclusion, PM2.5 induces inflammatory‐mediated MAPK pathway in artery which subsequently results in enhanced vascular contraction responding to 5‐HT via the upregulated 5‐HT2A receptors. [ABSTRACT FROM AUTHOR]

Background: The reported incidence of dengue fever increased dramatically in recent years in China. This study aimed to investigate and to assess the effectiveness of intervention implemented in a dengue outbreak in Ningbo City, Zhejiang Province, China. Methods: Data of a dengue outbreak were collected in Ningbo City in China by a field epidemiological survey according to a strict protocol and case definition. Serum specimens of all cases were collected for diagnosis and the virological characteristics were detected by using polymerase chain reaction (PCR) and gene sequencing. Vector surveillance was implemented during the outbreak to collect the larva and adult mosquito densities to calculate the Breteau Index (BI) and human biting rate (HBR), respectively. Data of monthly BI and light-trap density in 2018 were built to calculate the seasonality of the vector. A transmission mathematical model was developed to dynamic the incidence of the disease. The parameters of the model were estimated by the data of the outbreak and vector surveillance data in 2018. The effectiveness of the interventions implemented during the outbreak was assessed by the data and the modelling. Results: From 11 August to 8 September, 2018, a dengue outbreak was reported with 27 confirmed cases in a population of 5536-people community (community A) of Ningbo City. Whole E gene sequences were obtained from 24 cases and were confirmed as dengue virus type 1 (DENV-1). The transmission source of the outbreak was origin from community B where a dengue case having the same E gene sequence was onset on 30 July. Aedes albopictus was the only vector species in the area. The value of BI and HBR was 57.5 and 12 per person per hour respectively on 18 August, 2018 and decreased dramatically after interventions. The transmission model fitted well (χ2 = 6.324, P = 0.388) with the reported cases data. With no intervention, the total simulated number of the cases would be 1728 with a total attack rate (TAR) of 31.21% (95%CI: 29.99%– 32.43%). Case isolation and larva control (LC) have almost the same TAR and duration of outbreak (DO) as no intervention. Different levels of reducing HBR (rHBR) had different effectiveness with TARs ranging from 1.05% to 31.21% and DOs ranging from 27 days to 102 days. Adult vector control (AVC) had a very low TAR and DO. “LC+AVC” had a similar TAR and DO as that of AVC. “rHBR100%+LC”, “rHBR100%+AVC”, “rHBR100%+LC+AVC” and “rHBR100%+LC+AVC+Iso” had the same effectiveness. Conclusions: Without intervention, DENV-1 could be transmitted rapidly within a short period of time and leads to high attack rate in community in China. AVC or rHBR should be recommended as primary interventions to control rapid transmission of the dengue virus at the early stage of an outbreak. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi , an obligate intracellular pathogen, can infect various hosts. In this study, 3527 dairy cattle fecal specimens were collected from different geographic locations in China (including 673 from Shandong province, 1,440 from Guangdong province and 1,414 from Gansu province) and examined for the presence of E. bieneusi using polymerase chain reactions targeting the ribosomal internal transcribed spacer (ITS). The dominant genotypes identified were further subtyped by multilocus sequence typing. The overall prevalence of E. bieneusi was 14.2% (501/3527), with a significant difference in prevalence among the different geographical locations (P < 0.001). Our logistic regression analysis showed that all four variables (farming model, location, age, and clinical manifestations) had strong effects on the risk of contracting E. bieneusi. Sequence analysis revealed 11 genotypes: eight known genotypes (J, I, BEB4, BEB10, D, EbpC, CM19, and CM21) and three novel genotypes (named here as CGC1, CGC2, and CGC3). Genotypes J and I, the commonest, were found on all farms across the three provinces. Our linkage disequilibrium analysis showed a clonal population structure in the E. bieneusi dairy cattle population but the ITS genotypes had different population structures. Phylogenetic and haplotype network analysis showed the absence of geographical segregation in the E. bieneusi dairy cattle populations. Instead, they revealed the presence of host adaptation to the E. bieneusi populations in various animals. Our findings augment the current understanding of E. bieneusi transmission dynamics. [ABSTRACT FROM AUTHOR]

Background: Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Results: Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions: A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. [ABSTRACT FROM AUTHOR]

Background: Metastasis is a major threat to colorectal cancer (CRC) patients. We have reported that peroxiredoxin-2 (PRDX2) is associated with CRC invasion and metastasis. However, the mechanisms regulating PRDX2 expression remain unclear. We investigate whether microRNAs (miRNAs) regulate PRDX2 expression in CRC progression.Methods: Quantitative real-time polymerase chain reaction (qPCR) was used to measure microRNA-200b-3p (miR-200b-3p) expression. Immunohistochemistry (IHC) was performed to detect c-Myc and PRDX2 protein levels in CRC tissue samples (n = 97). Western blot was used to quantify PRDX2, c-Myc, AKT2/GSK3β pathway-associated proteins and epithelial-mesenchymal transition (EMT)-related proteins in CRC cells. Luciferase reporter assays were used to analyze the interaction between miR-200b-3p and 3'untranslated region (3'UTR) of PRDX2 mRNA and AKT2 mRNA as well as c-Myc and the miR-200b-3p promoter. Chromatin immunoprecipitation (ChIP) assay was used to evaluate binding of c-Myc to the miR-200b-3p promoter. Invasive assay and metastatic model were used to assess invasive and metastatic capacities of CRC cells in vitro and in vivo. Moreover, drug-induced apoptosis was measured by flow cytometry.Results: We found that miR-200b-3p was significantly downregulated, whereas c-Myc and PRDX2 were upregulated in metastatic CRC cells and CRC tissues compared to their counterparts. An inverse correlation existed between c-Myc and miR-200b-3p, and between miR-200b-3p and PRDX2. We also found that PRDX2 was a target of miR-200b-3p. Importantly, overexpression of nontargetable PRDX2 eliminated the suppressive effects of miR-200b-3p on proliferation, invasion, EMT, chemotherapeutic resistance and metastasis of CRC cells. Moreover, c-Myc bound to the promoter of miR-200b-3p and repressed its transcription. In turn, miR-200b-3p disrupted the stability of c-Myc protein by inducing c-Myc protein threonine 58 (T58) phosphorylation and serine 62 (S62) dephosphorylation via AKT2/GSK3β pathway.Conclusions: Our findings reveal that the c-Myc/miR-200b/PRDX2 loop regulates CRC progression and its disruption enhances tumor metastasis and chemotherapeutic resistance in CRC. [ABSTRACT FROM AUTHOR]

Anaerobic ammonium oxidation (anammox) as an important nitrogen removal pathway has been investigated in intertidal marshes. However, the rhizosphere-driven anammox process in these ecosystems is largely overlooked so far. In this study, the community dynamics and activities of anammox bacteria in the rhizosphere and non-rhizosphere sediments of salt-marsh grass Spartina alterniflora (a widely distributed plant in estuaries and intertidal ecosystems) were investigated using clone library analysis, quantitative PCR assay, and isotope-tracing technique. Phylogenetic analysis showed that anammox bacterial diversity was higher in the non-rhizosphere sediments ( Scalindua and Kuenenia) compared with the rhizosphere zone (only Scalindua genus). Higher abundance of anammox bacteria was detected in the rhizosphere (6.46 × 10-1.56 × 10 copies g), which was about 1.5-fold higher in comparison with that in the non-rhizosphere zone (4.22 × 10-1.12 × 10 copies g). Nitrogen isotope-tracing experiments indicated that the anammox process in the rhizosphere contributed to 12-14 % N generation with rates of 0.43-1.58 nmol N g h, while anammox activity in the non-rhizosphere zone contributed to only 4-7 % N production with significantly lower activities (0.28-0.83 nmol N g h). Overall, we propose that the rhizosphere microenvironment in intertidal marshes might provide a favorable niche for anammox bacteria and thus plays an important role in nitrogen cycling. [ABSTRACT FROM AUTHOR]

(B) Agarose gel electrophoresis (1.2%) exhibited a single and specific polymerase chain reaction product of each reference gene. Oncol Lett 14: 6001-6011, 2017; DOI: 10.3892/ol.2017.7002 Following the publication of the above article, the authors have realized that they inadvertently included the same data for the HSP90AB1 and ATP5B experiments shown in Fig. [Extracted from the article]

Abstract: Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS® and DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C T ) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C T values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 106 cells/sample or higher, which are levels not typically seen in ground beef. [Copyright &y& Elsevier]

The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province. [ABSTRACT FROM AUTHOR]

PSG076 is a pollen-specific gene isolated from wheat. The 1.4-kb promoter upstream of the ATG start codon was isolated by inverse-PCR (IPCR). To determine its activity, the PSG076 promoter was fused with the β-glucuronidase (GUS) reporter gene and introduced into tobacco. Histochemical analysis in transgenic tobacco showed that GUS activity was detected in late bicellular pollen grains and increased rapidly in mature pollen. GUS activity was also detected in pollen tubes of transgenic tobacco. No GUS activity was found in other floral and vegetable tissues. These results indicate that the PSG076 promoter directs pollen-specific activity at late stages of pollen development and pollen tube growth. Deletion analysis showed that a 0.4 kb fragment of the promoter was enough to confer pollen-specific expression. [ABSTRACT FROM AUTHOR]

Background and aims: To date, the association between special AT-rich sequence-binding protein 1 (SATB1) and colorectal cancer (CRC) has not been reported. This study was aimed at investigating the expression and potential role of SATB1 in human rectal cancers. Methods: Ninety-three paired samples of rectal cancer and distant normal rectal tissue were analyzed by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), and the correlations between SATB1 expression and clinicopathological parameters were evaluated. The expression profiles of SATB1 were also investigated in a panel of five human colon carcinoma cell lines. Results: The general level of SATB1 mRNA in rectal cancer tissues was statistically significantly higher than that in normal mucosa ( P = 0.043). The rate of positive SATB1 protein expression in rectal cancers (44.1%) was significantly higher than that in normal tissues (25.8%) by IHC analysis ( P = 0.009). Overexpression of SATB1 mRNA was more predominant in patients with earlier onset of rectal cancer ( P = 0.033). SATB1 expression correlated with invasive depth and tumor node metastasis (TNM) stage at both protein and mRNA levels ( P < 0.05). Furthermore, SATB1 expression in the poorly metastatic KM12C cells was significantly lower than the highly metastatic KM12SM and KM12L4A cells and higher than the HCT116 and SW480 cells ( P = 0.001). These results were further confirmed by Western blotting. Conclusion: Our results indicate that SATB1 may play an important role in the progression of human rectal cancer, which represents a possible new mechanism underlying CRC. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effects of ardipusilloside I isolated from Ardisia pusilla on tumor angiogenesis and its mechanism of action. The anti-angiogenic effect in vivo was evaluated on xenograft in the athymic mice model and the chicken chorioallantoic membrane (CAM) neovascularization model, the inhibition of growth in vitro was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the mechanism was demonstrated through detecting microvessel density (MVD), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and P-VEGFR2 protein expressions, as well as mRNA expressions of VEGF and VEGFR2. The results showed that ardipusilloside I had a good inhibitory effect on A549 xenografted tumor growth, angiogenesis of CAM, and A549 cell growth. Compared to the negative control, MVD protein and mRNA expressions of VEGF and VEGFR were significantly inhibited by ardipusilloside I in a dose-dependent manner. These findings suggested that ardipusilloside I might be a promising candidate as angiogenesis inhibitors. [ABSTRACT FROM AUTHOR]

Adiponectin is an adipose-derived hormone that has anti-diabetic and anti-atherogenic effects through interaction with AdipoR1 and AdipoR2 (adiponectin receptors 1 and 2), but little is known about the expression and function of adiponectin and its receptors in adventitia and adventitial fibroblasts. In the present study, we have demonstrated that AdipoR1 is highly expressed in rat adventitia and cultured adventitial fibroblasts by quantitative real-time PCR, Western blotting and immunofluorescent staining, whereas Adipo2 is low-expressed. The expression of AdipoR1 have been observed to decrease gradually in adventitial fibroblasts in response to LPS (lipopolysaccharide) treatment. No local expression of adiponectin has been detected in adventitial tissues, indicating that serum adiponectin is the ligand for AdipoR1 in adventitial fibroblasts. In addition, treatment of recombinant adiponectin inhibited LPS-induced proliferation of adventitial fibroblasts via activation of the AMPK (adenosine monophosphate-activated protein kinase). AdipoR1 siRNA (small interfering RNA) transfection potently knocked down the receptor protein. The siRNA-AdipoR1 transfected cells and AMPK inhibitor compound C treated cells showed decreased phosphorylated level of AMPK as determined by Western blot analysis, and increased the proliferation of adventitial fibroblasts as determined by BrdU (5-bromo-29-deoxyuridine) staining. These results demonstrated that adiponectin stimulates the proliferation of adventitial fibroblasts via the AdipoR1 and AMPK signalling pathways. [ABSTRACT FROM AUTHOR]

Background: Most current animal models of hindlimb ischemia use acute arterial occlusion that does not accurately reflect the pathogenesis of gradual arterial occlusion in humans. We, therefore, developed the first mouse model of gradual arterial occlusion and tested the hypothesis that the mechanisms regulating blood flow recovery are critically dependent on the rate of arterial occlusion. Methods: Gradual arterial occlusion was induced by placing ameroid constrictors on the proximal and distal left femoral artery, and ligating the femoral arterial branches (n = 36). Acute arterial occlusion was accomplished by excising the left femoral artery (n = 36). The blood flow recovery was studied by laser Doppler imaging. Differential gene expression between these two models was assessed by quantitative real-time polymerase chain reactions (PCR). Inflammatory and progenitor cells recruitment were determined by immunohistochemistry. Results: We found that hypoxia-related genes increased significantly in the calf, but not in the thigh, after gradual and acute femoral arterial occlusion (P < .05). Shear-stress dependent genes and inflammatory genes were upregulated immediately in the thigh only after acute femoral arterial occlusion (P < .05). These differences in gene expression were consistent with increased SDF-1α expression, recruitment of macrophages and hemangiocytes, and higher blood flow recovery after acute arterial occlusion than after gradual arterial occlusion (P < .05). Conclusion: This is the first study to show the mechanisms that regulate blood flow recovery are critically dependent on the rate of arterial occlusion. This novel model of gradual arterial occlusion may more closely resemble the human diseases, and may provide more accurate mechanistic insights for creating novel molecular therapies. [Copyright &y& Elsevier]

Guillain–Barre Syndrome (GBS) is a neuromuscular disorder and campylobacteriosis is known to trigger the onset of the disorder. A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 497-bp region of the UDP-galactose 4-epimerase (galE) gene sequence in campylobacters responsible for triggering the onset of GBS. The identity of the PCR product was confirmed by HindIII endonuclease restriction digestion, which produced the predicted 430 and 67-bp DNA fragments. The assay could detect the presence of the gene in Campylobacter suspensions containing as few as 5 cells ml−1. The assay detected the presence of the gene in 17 of the 20 campylobacters isolated from chicken, 9 of the 13 campylobacters isolated from turkey and 7 of the 7 campylobacters isolated from human stools. All Campylobacter strains isolated from chicken, turkey and clinical samples were resistant to multiple antibiotics. The assay failed to detect the presence of the gene in five different microaerophilic strains of Helicobacter spp., E. coli and Salmonella spp. The entire diagnostic assay, including template preparation, amplification and electrophoresis, can be completed within 6 h. [Copyright &y& Elsevier]

An oligonucleotide-microarray method was developed for the detection of intestinal bacteria in fecal samples collected from human subjects. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to glass slides. Cyanine5 (CY5)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or bacterial DNA using two universal primers and were hybridized to the oligo-microarray. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides fragilis, Bacteroides distasonis, Clostridium clostridiiforme, Clostridium leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus albus, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis, Eubacterium biforme, Eubacterium aerofaciens, Lactobacillus acidophilus, Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the oligo-microarray method developed in this study is a reliable method for the detection of predominant human intestinal bacteria in the fecal samples. [Copyright &y& Elsevier]

Outbreaks of Vibrio parahaemolyticus gastroenteritis in the United States (Texas, New York and Pacific Northwest) in 1997–98 emphasized the need to develop molecular methods for identification and differentiation of these organisms. When outbreak isolates were analyzed for the enterobacterial repetitive intergenic consensus sequences, the Texas and New York outbreak isolates had a specific 850-bp DNA fragment that was absent in Pacific Northwest isolates. The 850-bp polymerase chain reaction (PCR) product was found in isolates of serovar O3:K6, which have an unusual potential to spread and cause infections. To develop a specific molecular detection method for serovar O3:K6, the nucleotide sequence of the 850-bp product was determined. The GenBank blast analysis did not show homology with any known Vibrio spp. gene sequences. Two PCR primers were designed to specifically amplify the unique sequences from serovar O3:K6 isolates. Genomic DNA from 10 Texas, eight New York, and seven Pacific Northwest outbreak isolates of V. parahaemolyticus was assayed by PCR. Texas and New York isolates were positive in the PCR assay, giving a 327-bp PCR product as predicted; however, Pacific Northwest isolates were negative, indicating the absence of the target gene. Texas and New York isolates were all serovar O3:K6; the Pacific Northwest isolates were not. The primers were tested with other Vibrio spp. and other closely related species and no amplification of the 327-bp PCR product was found. The PCR method can be used to specifically identify O3:K6 V. parahaemolyticus isolates in less than 6 h. [Copyright &y& Elsevier]

Reports on the development of polymerase chain reaction (PCR) methods based on the 16S rRNA genes of Mycobacterium sp. PYR-1 and Mycobacterium sp. PAH135. Polycyclic aromatic hydrocarbons (PAH) mineralization; PCR products confirmation by a restriction enzyme digestion.

Objectives: MicroRNAs possess essential effects on gastric cancer (GC), whereas the underlying mechanisms have not been fully uncovered. The present work focused on investigating the role of miR-381-3p in GC cellular processes and the possible mechanisms. Materials and Methods: miR-381-3p levels within GC tissues and cells were measured through quantitative real-time polymerase chain reaction (qRT-PCR). This study measured cell proliferation, apoptosis, and metastasis through EdU, colony formation, flow cytometry, and Transwell assays separately. TargetScan was adopted to predict the miR-381-3p targets, whereas luciferase reporter assay was adopted for confirmation. Results: miR-381-3p levels were decreased, whereas fibroblast growth factor receptor-2 (FGFR2) expression was increased in GC. miR-381-3p upregulation inhibited proliferation, migration, and invasion and it promoted the apoptosis of GC cells. Further, FGFR2 overexpression partly reversed the miR-381-3p-mediated impacts on GC cellular processes. Conclusions: This study provides an experimental basis, suggesting the potential of using miR-381-3p as the novel marker for GC. Clinical Trial Registration number: 2020-05. [ABSTRACT FROM AUTHOR]

Blastocystis is one of the important zoonotic parasites which can infect humans and various animals worldwide and has become a growing global public health concern. The study aims to obtain the data of Blastocystis infection and the information of the genetic characteristic. In the present study, 489 fecal samples were collected from diarrhea outpatients in Ningbo, Zhejiang province, and were examined the presence of Blastocystis by polymerase chain reaction combined with sequencing. A total of 10 samples (2.04%, 10/489) were positive for Blastocystis with no significant difference among sex and age groups, respectively. Eight samples were successfully sequenced, and five zoonotic ST3 and three zoonotic ST1 with two new sequences were identified. We first demonstrated the occurrence of Blastocystis infection in diarrhea outpatients in Ningbo, with two zoonotic subtypes (ST1 and ST3) and two new sequences being characterized. Meanwhile, mixed infection of Blastocystis and E. bieneusi was found which indicates the importance of investigation of multiple parasites. Finally, more extensive studies will be needed to better understand the transmission of Blastocystis at human-animal-environment interface and provide evidence for the development of one health strategies for the prevention and control of such diseases. • Blastocystis infection in diarrhea outpatients first reported in Ningbo. • Zoonotic subtypes of ST1 and ST3 and two new sequences characterized. • Mixed infection of Blastocystis and E. bieneusi identified. [ABSTRACT FROM AUTHOR]

Livestock operations are known to harbor elevated levels of antibiotic resistance genes (ARGs) that may pose a threat to public health. Broiler feedlots may represent an important source of ARGs in the environment. However, the prevalence and dissemination mechanisms of various types of ARGs in the environment of broiler feedlots have not previously been identified. We examined the occurrence, abundance and variation of ARGs conferring resistance to chloramphenicols, sulfonamides and tetracyclines in the environments of two representative types of broiler feedlots (free range and indoor) by quantitative PCR, and assessed their dissemination mechanisms. The results showed the prevalence of various types of ARGs in the environmental samples of the broiler feedlots including manure/litter, soil, sediment, and water samples, with the first report of five chloramphenicol resistance genes (cmlA, floR, fexA, cfr, and fexB) in broiler feedlots. Overall, chloramphenicol resistance genes and sulfonamides sul genes were more abundant than tetracyclines tet genes. The ARG abundances in the samples from indoor boiler feedlots were generally different to the free range feedlots, suggesting the importance of feeding operations in ARG dissemination. Pearson correlation analysis showed significant correlations between ARGs and mobile genetic element genes (int1 and int2), and between the different classes of ARGs themselves, revealing the roles of horizontal gene transfer and coselection for ARG dissemination in the environment. Further regression analysis revealed that fexA, sul1 and tetW could be reliable indicator genes to surrogate anthropogenic sources of ARGs in boiler feedlots (correlations of fexA, sul1 and tetW to all ARGs: R = 0.95, 0.96 and 0.86, p < 0.01). Mean while, significant correlations were also identified between indicator ARGs and their corresponding antibiotics. In addition, some ARGs were significantly correlated with typical metals (e.g., Cu, Zn, and As with fexA, fexB, cfr, sul1, tetW, tetO, tetS: R = 0.52-0.71) and some environmental parameters (e.g., TOC, TN, TP, NH3-N with fexA, fexB, cfr, sul1, tetW, tetO, tetQ, tetS: R = 0.53-0.87) (p < 0.01). Further redundancy analysis demonstrated that the distribution and transportation of ARGs from the boiler feedlots to the receiving environments were correlated with environmental variables. The findings highlight the contribution of some chemicals such as antibiotics and metals to the development of ARGs in broiler feedlots environments; and the observed ARG dissemination mechanism in the broiler feedlots facilitates the development of effective mitigation measures. [ABSTRACT FROM AUTHOR]