Absolute Quantitative Real-time PCR Research Articles

Characterization of the Effects of Host p53 and Fos on Gallid Alpha Herpesvirus 1 Replication.

Treatment options for herpesvirus infections that target the interactions between the virus and the host have been identified as promising. Our previous studies have shown that transcription factors p53 and Fos are essential host determinants of gallid alpha herpesvirus 1 (ILTV) infection. The impact of p53 and Fos on ILTV replication has 'not been fully understood yet. Using the sole ILTV-permissive chicken cell line LMH as a model, we examined the effects of hosts p53 and Fos on all phases of ILTV replication, including viral gene transcription, viral genome replication, and infectious virion generation. We achieved this by manipulating the expression of p53 and Fos in LMH cells. Our results demonstrate that the overexpression of either p53 or Fos can promote viral gene transcription at all stages of the temporal cascade of ILTV gene expression, viral genome replication, and infectious virion production, as assessed through absolute quantitative real-time PCR, ILTV-specific RT-qPCR assays, and TCID50 assays. These findings are consistent with our previous analyses of the effects of Fos and p53 knockdowns on virus production and also suggest that both p53 and Fos may be dispensable for ILTV replication. Based on the synergistic effect of regulating ILTV, we further found that there is an interaction between p53 and Fos. Interestingly, we found that p53 also has targeted sites upstream of ICP4, and these sites are very close to the Fos sites. In conclusion, our research offers an in-depth understanding of how hosts p53 and Fos affect ILTV replication. Understanding the processes by which p53 and Fos regulate ILTV infection will be improved by this knowledge, potentially paving the way for the development of novel therapeutics targeting virus-host interactions as a means of treating herpesvirus infections.

In vivo and in silico investigations on the efficacy of albendazole against Enterocytozoon hepatopenaei (EHP) infecting Penaeus vannamei

The microsporidian Enterocytozoon hepatopenaei (EHP) is an intercellular spore former that causes hepatopancreatic microsporidiosis (HPM), a commercially important disease in Penaeus vannamei (white leg shrimp) farming. In this study, albendazole an inhibitor of β-tubulin polymerization in microsporidians was evaluated for its efficacy in reducing EHP infection in P. vannamei. EHP-infected shrimps were exposed to 1, 5, 10, 25, 50 and 75 mg L−1 of albendazole and evaluated for the reduction in EHP DNA copy numbers at 3, 6, 9, 12, 15, 18, 21, 24 days post-exposure (dpe) by absolute quantitative real-time PCR (RT-qPCR) assay. The expressions of antioxidant enzyme genes viz., cytosolic manganese superoxide dismutase (cMnSOD), catalase (CAT) and glutathione peroxidase (GPx); haemolymph aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were assessed at 24 dpe. A maximum of about 98% reduction in EHP DNA copies was observed in the shrimps treated with 75 mg L−1 per day for 24 days. A significant upregulation in relative expressions of GPx genes and a downregulation cMnSOD and CAT genes were observed in shrimps exposed to 25, 50 and 75 mg L‐1 at 24 dpe. A significant reduction in haemolymph AST and ALT levels was observed upon treatment with 25, 50 and 75 mg L−1 at 24 dpe and there are no significant changes in ALP. A virtual molecular docking between albendazole and EHP encoded β-tubulin revealed a best binding interaction with the presence of 11 hydrogens bonds, multiple non-covalent bonds and a very much lower solvation-free energy gain of −15.6 kcal mol−1 in the active sites of protein interactions. Thus, this study identified albendazole as a potential drug to control EHP infection in shrimp, which might help to combat the production losses caused by EHP.

Actin depolymerizing factor-based nanomaterials: A novel strategy to enhance E. mitis-specific immunity.

The epidemic of avian coccidiosis seriously threatens the animals' welfare and the economic gains of the poultry industry. Widespread in avian coccidiosis, Eimeria mitis (E. mitis) could obviously impair the production performance of the infected chickens. So far, few effective vaccines targeting E. mitis have been reported, and the nanovaccines composed of nanospheres captured our particular attention. At the present study, we construct two kinds of nanospheres carrying the recombinant E. mitis actin depolymerizing factor (rEmADF), then the characterization was then analyzed. After safety evaluation, the protective efficacy of rEmADF along with its nanospheres were investigated in chickens. The promoted secretions of antibodies and cytokines, as well as the enhanced percentages of CD4+ and CD8+ T cells were evaluated by the ELISA and flow cytometry assay. In addition, the absolute quantitative real-time PCR (qPCR) assay implied that vaccinations with rEmADF-entrapped nanospheres could significantly reduce the replications of E. mitis in feces. Compared with the rEmADF-loaded chitosan (EmADF-CS) nanospheres, the PLGA nanospheres carrying rEmADF (EmADF-PLGA nanosphers) were more effective in up-regulating weight efficiency of animals and generated equally ability in controlling E. mitis burdens in feces, suggesting the PLGA and CS nanospheres loaded with rEmADF were the satisfactory nanovaccines for E. mitis defense. Collectively, nanomaterials may be an effective antigen delivery system that could help recombinant E. mitis actin depolymerizing factor to enhance immunoprotections in chicken against the infections of E. mitis.

Microbiota-host crosstalk in the newborn and adult rumen at single-cell resolution

BackgroundThe rumen is the hallmark organ of ruminants, playing a vital role in their nutrition and providing products for humans. In newborn suckling ruminants milk bypasses the rumen, while in adults this first chamber of the forestomach has developed to become the principal site of microbial fermentation of plant fibers. With the advent of single-cell transcriptomics, it is now possible to study the underlying cell composition of rumen tissues and investigate how this relates the development of mutualistic symbiosis between the rumen and its epithelium-attached microbes.ResultsWe constructed a comprehensive cell landscape of the rumen epithelium, based on single-cell RNA sequencing of 49,689 high-quality single cells from newborn and adult rumen tissues. Our single-cell analysis identified six immune cell subtypes and seventeen non-immune cell subtypes of the rumen. On performing cross-species analysis of orthologous genes expressed in epithelial cells of cattle rumen and the human stomach and skin, we observed that the species difference overrides any cross-species cell-type similarity. Comparing adult with newborn cattle samples, we found fewer epithelial cell subtypes and more abundant immune cells, dominated by T helper type 17 cells in the rumen tissue of adult cattle. In newborns, there were more fibroblasts and myofibroblasts, an IGFBP3+ epithelial cell subtype not seen in adults, while dendritic cells were the most prevalent immune cell subtype. Metabolism-related functions and the oxidation-reduction process were significantly upregulated in adult rumen epithelial cells. Using 16S rDNA sequencing, fluorescence in situ hybridization, and absolute quantitative real-time PCR, we found that epithelial Desulfovibrio was significantly enriched in the adult cattle. Integrating the microbiome and metabolome analysis of rumen tissues revealed a high co-occurrence probability of Desulfovibrio with pyridoxal in the adult cattle compared with newborn ones while the scRNA-seq data indicated a stronger ability of pyroxidal binding in the adult rumen epithelial cell subtypes. These findings indicate that Desulfovibrio and pyridoxal likely play important roles in maintaining redox balance in the adult rumen.ConclusionsOur integrated multi-omics analysis provides novel insights into rumen development and function and may facilitate the future precision improvement of rumen function and milk/meat production in cattle.

Evaluating the predictive performance of gut microbiota for the early-stage colorectal cancer

BackgroundColorectal cancer (CRC) has been regarded as one of the most frequently diagnosed malignancies among the leading causes of cancer-related morbidity and mortality globally. Diagnosis of CRC at the early-stages of tumour might improve the survival rate of patients. The current study sought to determine the performance of fecal Fusobacterium nucleatum (F. nucleatum) and Streptococcus bovis (S. bovis) for timely predicting CRC.MethodsThrough a case–control study, the fecal sample information of 83 individuals (38 females, 45 males) referring to a hospital in Tehran, Iran was used. All patients underwent a complete colonoscopy, regarded as a gold standard test. Bacterial species including S. bovis and F. nucleatum were measured by absolute quantitative real-time PCR. The Bayesian univariate and bivariate latent class models (LCMs) were applied to estimate the ability of the candidate bacterial markers in order to early detection of patients with CRC.ResultsBayesian univariate LCMs demonstrated that the sensitivities of S. bovis and F. nucleatum were estimated to be 86% [95% credible interval (CrI) 0.82–0.91] and 82% (95% CrI 0.75–0.88); while specificities were 84% (95% CrI 0.78–0.89) and 80% (95% CrI 0.73–0.87), respectively. Moreover, the area under the receiver operating characteristic curves (AUCs) were 0.88 (95% CrI 0.83–0.94) and 0.80 (95% CrI 0.73–0.85) respectively for S. bovis and F. nucleatum. Based on the Bayesian bivariate LCMs, the sensitivities of S. bovis and F. nucleatum were calculated as 93% (95% CrI 0.84–0.98) and 90% (95% CrI 0.85–0.97), the specificities were 88% (95% CrI 0.78–0.93) and 87% (95% CrI 0.79–0.94); and the AUCs were 0.91 (95% CrI 0.83–0.99) and 0.88(95% CrI 0.81–0.96), respectively.ConclusionsOur data has identified that according to the Bayesian bivariate LCM, S. bovis and F. nucleatum had a more significant predictive accuracy compared with the univariate model. In summary, these intestinal bacteria have been highlighted as novel tools for early-stage CRC diagnosis.

Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail.

Quantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse transcription methods. In the experiments, we focused on the cerebral giant cell (CGC), a key neuron required for the acquisition of conditioned taste aversion in the pond snail Lymnaea stagnalis, and examined changes in the mRNA levels of 3 memory-related genes, cAMP-response element binding proteins (LymCREB1 and LymCREB2) and CREB-binding protein (LymCBP), during memory formation. The results obtained by relative quantification showed similar patterns for the 3 genes. For absolute quantification, reverse transcription was performed using 2 different methods: a mixture of oligo d(T) primers and random primers (RT method 1); and gene-specific primers (RT method 2). These methods yielded different results and did not show consistent changes related to conditioning. The mRNA levels in the samples prepared by RT method 2 were up to 3.3 times higher than those in samples prepared by RT method 1. These results suggest that for qPCR of single neurons, the efficacy and validity do not differ between relative and absolute quantification methods, but the reverse transcription step critically influences the results of mRNA quantification.

Impact of Nutritional Profile on Gut Microbiota Diversity in Patients with Celiac Disease

The pathogenesis of celiac disease (CD) is significantly influenced by gut microbiota. Daily nutritional profile influences the diversity of gut microbiota. This study was aimed to compare the abundance of gut microbiota in CD patients compared to normal control (NC), and to investigate the impact of nutritional factors on their fecal microbiota diversity. In this study, a selected panel of intestinal bacteria was assessed in 31 confirmed CD patients adhering to gluten-free diet (GFD) for more than 6months and in 20 NC subjects. Stool samples were collected from each participant, DNA was extracted, and absolute quantitative real-time PCR (qPCR) was carried out. The gut microbiota including Bacteroidetes, Bifidobacterium, Clostridium, Staphylococcus, Enterobacteiaceae, Firmicutes, and Lactobacillus were assessed. The quantities of fruits, vegetables, meat, liquids, sugar and gluten-free candy/bread consumption were evaluated using a questionnaire. The proportion of Bifidobacterium, Firmicutes, and Lactobacillus in CD cases was significantly lower than NC (P < 0.005). Significant correlation coefficients between Bifidobacterium and Lactobacillus (P < 0.001), and also Firmicutes and Lactobacillus (P < 0.001) were recorded. Moreover, a significant association between medium amount of meat and bean consumptions and low abundance of Lactobacillus and Firmicutes (P = 0.024 and P = 0.027, respectively), and also high amount of bean consumptions and low abundance of Lactobacillus (P = 0.027) in CD were observed. The results showed that meat and bean consumptions could reduce the beneficial bacteria including Firmicutes and Lactobacillus in CD patients. Therefore, changes in the gut microbiota abundance may contribute to dietary changes and unimproved CD symptoms.

Streptococcus mutans-associated bacteria in dental plaque of severe early childhood caries

ABSTRACT Background Streptococcus mutans (S. mutans) is a potential pathogenic bacteria of dental caries. However, the level of S. mutans is low in some children with severe early childhood caries (SECC) Aim To evaluate the effect of S. mutans level on dental microbiome and cariogenesis. Methods The oral microbiota was compared between caries-free group (CF) and SECC group.16S rRNA gene sequencing was used for S. mutans level bacterial community analysis. The candidate bacteria that were closely related with S. mutans abundance were identified and confirmed by absolute quantitative real-time PCR in clinical dental plaque samples from CF and SECC groups. Results Through in-depth analysis of dental plaque microorganism, Leptotrichia, Selenomonas and Prevotella_7 were found in the S. mutans-low group (p < 0.05) and Porphyromonas, Selenomonas_3 were found in the S. mutans-high group (p < 0.05). Through quantitative real-time PCR, Leptotrichia, Selenomonas and Prevotella_7 were identified as the potential biomarkers of SECC when S. mutans was at a low level. Conclusion Leptotrichia, Selenomonas and Prevotella_7 are identified as potential biomarkers in SECC with a low abundance or without S. mutans. Our study may shed light on the understanding of caries occurrence in SECC with low abundance of S. mutans. Abbreviations S. mutans, Streptococcus mutans; CF, caries-free; SECC, severe early childhood caries; ECC, early childhood caries; rRNA, ribosome RNA; qPCR, Quantitative real-time PCR; OTUs, operational taxonomic units; ANOVA, analysis of variance; LDA, Linear discriminant analysis; LEfSe, Linear discriminant analysis effect size; COG, Groups of proteins; NMDS, Non-MetricMulti-Dimensional Scaling; IL-1β, interleukin −1β; IL-6, interleukin-6; IL-8, interleukin-8; IL-10, interleukin-10.

Detection of Yeast-like Symbionts in Brown Planthopper Reared on Different Resistant Rice Varieties Combining DGGE and Absolute Quantitative Real-Time PCR.

Simple SummaryThe brown planthopper (BPH) is an important pest that causes huge losses in rice production. The promotion and use of insect-resistant rice varieties is an important way to control BPH. However, in practice, BPH can adapt to resistant rice within several generations. Endosymbionts may be one of the reasons for the rapid adaptation of BPH to resistant rice. The BPH harbor yeast-like symbionts (YLS) in their abdomen, and YLS are essential for the nutrition, development, and reproduction of BPH. Our previous report showed that among the YLS communities detected in BPH, Ascomycetes symbionts, Pichia-like symbionts, and Candida-like symbionts were the three dominant populations of YLS. In this study, PCR-DGGE and absolute quantitative real-time PCR were used to detect the variations of three dominant YLS in BPH across different nymph ages and on different resistant rice varieties. The results showed that the total number of YLS gradually increased from the first instar to adulthood, but decreased in the fifth instar nymph, when BPH were reared on the susceptible rice variety TN1. The rice-resistant varieties, Mudgo, ASD7, and RH have more significant inhibitory effects on the three dominant YLS in the first and second generations of BPH. However, the numbers of the three dominant YLS were all recovered from the third generation of BPH. Ascomycetes symbionts were the most dominant strain among the three YLS.The brown planthopper (BPH), Nilaparvata lugens, is a serious pest of rice throughout Asia. Yeast-like symbionts (YLS) are endosymbionts closely linked with the development of BPH and the adapted mechanism of BPH virulence to resistant plants. In this study, we used semi-quantitative DGGE and absolute quantitative real-time PCR (qPCR) to quantify the number of the three YLS strains (Ascomycetes symbionts, Pichia-like symbionts, and Candida-like symbionts) that typically infect BPH in the nymphal stages and in newly emerged female adults. The quantities of each of the three YLS assessed increased in tandem with the developing nymphal instar stages, peaking at the fourth instar stage, and then declined significantly at the fifth instar stage. However, the amount of YLS present recovered sharply within the emerging adult females. Additionally, we estimated the quantities of YLS for up to eight generations after their inoculation onto resistant cultivars (Mudgo, ASD7, and RH) to reassociate the dynamics of YLS with the fitness of BPH. The minimum number of each YLS was detected in the second generation and gradually increased from the third generation with regard to resistant rice varieties. In addition, the Ascomycetes symbionts of YLS were found to be the most abundant of the three YLS strains tested for all of the development stages of BPH.

Development of a subunit vaccine based on fiber2 and hexon against fowl adenovirus serotype 4

Hepatitis-hydropericardium syndrome (HHS) is widespread in China and causes high chicken mortality that results in great economic losses. A safe and effective vaccine is needed, and a subunit vaccine has potential for development. In this study, a truncated region of the FAdV-4 fiber 2 fused with coding sequence of one epitope of hexon was expressed in a prokaryotic expression system, and the immune protective effects of different doses of recombinant fiber 2 subunit vaccine on SPF chickens were compared. The recombinant fiber2 (Gly275- Pro479 aa)-hexon (Met21-Val51 aa) protein (rFH) obtained in Escherichia coli showed good solubility. The chicken survival rate at the lowest dose (2.5 μg/bird) was 75% (6/8), and at higher doses (≥5 μg/bird) was 100% (8/8) in challenge experiment. Two chickens in the 2.5 μg/bird treatment showed severe lesions, while birds in the higher dose treatments showed no obvious tissue damage as determined by histopathologic analysis of liver and spleen. Absolute quantitative real-time PCR showed no viral load in the ≥5 μg/bird treatments, but two chickens in the 2.5 μg/bird treatment had high viral loads. The challenge experience demonstrated that the rFH vaccine provided 100% protection at ≥5 μg/bird. These results suggested that rFH protein as an effective vaccine to protect against FAdV-4 and provided a new idea for the development of vaccine against HHS.

Quantification of circadian rhythm in mitochondrial DNA copy number in whole blood, and identification of factors that influence it

Mitochondrial DNA (mtDNA), the genetic material in mitochondria, encodes key genes related to the respiratory chain and ATP production. To accurate quantification mtDNA content in whole blood is important for various disease states. Absolute quantitative Real-time PCR and platelet contamination erase method were used for mtDNA copy number analysis in whole blood. In the quantitative study of mtDNA content, it was found that whole blood mtDNA copy number showed a fluctuating rhythm during a 24-h period due to dynamic changes in white blood cells combined with platelets. However, when isolated white blood cells were used, or absolute whole blood mtDNA was calculated, the circadian rhythm pattern of mtDNA disappeared. In this study, a feasible method that can accurately quantify mitochondrial DNA in small blood samples was established, and it was found that two factors which greatly influenced mtDNA copy number were sampling time and platelets in blood.

Novel Biomarkers Aim at Detecting Metastatic Sentinel Lymph Nodes in Breast Cancer

Background:Intra-operative molecular diagnostic assays are currently used for the detection of lymph node metastases. The objective of this study was to find new biomarkers to improve diagnostic accuracy in the detection of metastatic axillary lymph nodes in breast cancer patients. Methods:We applied an absolute quantitative real-time RT-PCR to quantitate the expression of CK19, KLK11, and CLEC3A mRNAs in 79 FFPE SLNs from 35 breast cancer patients. The CK19 was confirmed as a standard biomarker, and the level of expression of selected new markers, KLK11 and CLEC3A, was evaluated in pathologically negative and positive SLNs by using absolute quantitative real-time PCR. Results:The overall concordance of the CK19 gene with pathological results was 92.4% (less than 250 copies) in negative SLNs and 85% in positive SLNs (more than 250 copies). The sensitivity and specificity of CK19, which were detected by real-time PCR, was 85% and 46%, respectively. Our results revealed that lower CLEC3A was associated with more lymph node involvement. We could set a cut-off point for CLEC3A with the sensitivity of 78% and specificity of 60%. Also, the mean KLK11 had a statistically significant reverse correlation with tumor grade (p = 0.017). Higher CK19 levels were related to more tumor invasion (p < 0.0001). Conclusion:Regarding the findings, CLEC3A along with CK19 can be used as a promising marker with high sensitivity and specificity for the detection of metastatic SLN.

High frequency of Chlamydia pneumoniae and risk factors in children with acute respiratory infection.

This study was performed as a contribution for a better understanding of Chlamydia pneumoniae frequency in children with respiratory infections. A total of 416 children were recruited from two clinical centers in Sao Luis, Brazil. Of these patients, 165 children had upper respiratory tract infections (URTI), 150 had community-acquired pneumonia (CAP), and 101 were asymptomatic volunteer children. Clinical and epidemiological data from the participants were recorded. Nasopharyngeal swab samples were collected to extract DNA. C. pneumoniae DNA positivity and copy numbers were obtained by an absolute quantitative real-time PCR method. RESULTS: Positivity for C. pneumoniae DNA was higher in samples from URTI children (38.2%) and from CAP children (18.0%) than in those from the control group (7.9%; p < 0.001). Moreover, C. pneumoniae DNA was denser in children with URTI than in asymptomatic children. Considering the cutoff, the highest value of C. pneumoniae DNA found in asymptomatic children of the 3.98 log10 copies/mL, 8.5% (14/165) of the children with URTI, and 3.3% (5/150) with CAP presented high copy numbers of C. pneumoniae DNA. CONCLUSION: Taken together, these results revealed a high frequency of C. pneumoniae in both children with URTI and CAP.

Effect of Estradiol on miR-21&amp; miR-155 Expression in promyelocytic leukemia-derived cell line NB4

Background: Due to the estrogen participation in modulating the proliferation and commitment of stem cells and the effects of miR-21 and miR-155 expression on reduced proliferation and colony formation of acute myeloid leukemia (AML), the aim of the present study was to evaluate the effect of estradiol on expression of miR-21 and miR-155 in the NB4 cell line, as an acute promyelocytic leukemia (APL).&#x0D; Materials and Methods: In the present experiment, NB4 cells were treated with different quantities of estradiol (5, 25, 50, 75, 100, 150, 200, 250 μg/ml) and vehicle control for 24 and 48 hours. Viability, apoptosis, and cellular proliferation were estimated by trypan blue exclusion, flow cytometry, and MTT assays, respectively. The level of miR-155 and miR-21 expression was studied using absolute quantitative real-time PCR.&#x0D; Results: Results showed that estradiol in the effective dose (200 μg/ml) led to decreased cellular viability (in a dose dependent manner, P = 0.004) and apoptosis of NB4 cells. In addition, the expressions of miR-155 and miR-21 were significantly and dose-dependently decreased (p&lt;0.05).&#x0D; Conclusion: Estradiol at the effective dose caused apoptosis in NB4 cell line. This substance can be used as a drug for the treatment of APL. However, further assessments are needed to support the effectiveness of estradiol in the treatment of APL.

Age-related intestinal monosaccharides transporters expression and villus surface area increase in broiler and layer chickens.

In the chicken small intestine, glucose is mainly transported by the apically located sodium/glucose cotransporter 1 (SGLT1) and the basolaterally located glucose transporter 2 (GLUT2). Fructose is transported by the apically located glucose transporter 5 (GLUT5) and similarly by GLUT2. During the early post-hatching period, the intestinal villus surface area (VSA) should be considered as an important factor related to the monosaccharide absorption capacity. Our objective here was to study intestinal monosaccharide absorption by analyzing the effects of age, diet, and breed on monosaccharide transporters and the VSA. The mRNA expression patterns of SGLT1, GLUT2 and GLUT5 genes in broiler and layer chickens were measured from the day of hatching to day 28 using the absolute quantitative real-time PCR. Both the intestinal mRNA expression levels of these genes and the VSA were affected by age. The mRNA expression levels of SGLT1 and GLUT2 were significantly increased from day 1 to day 3 and then decreased from day 3 to day 28. The expression levels of GLUT5 decreased from day 1 to day 7. The broiler chickens VSAs were significantly larger than those of the layer chickens from days 7 to 28. The effect of diet on the gene expression patterns of these monosaccharide transporters and the VSA were not significant. Our results suggest that the expression levels of these monosaccharide transporters are increased rapidly at the beginning of intestinal growth to meet the demands for monosaccharides to support the fast growth of the chick before day 7. As intestinal maturation and VSA increased, the expression levels of these monosaccharide genes decreased to a certain expression level to maintain the intestinal transport capacity and the absorption balance of all other nutrients.

Applying simple linear combination, multiple logistic and factor analysis methods for candidate fecal bacteria as novel biomarkers for early detection of adenomatous polyps and colon cancer

BackgroundColorectal cancer (CRC) is the third leading cause of cancer, and presents a considerable disease burden, worldwide. Recently, the gut microbiota has been proposed as a potential risk factor for CRC, and even adenomatous polyps (AP). Here, the aim of this study was to investigate the role of selected gut bacteria as fecal bacterial biomarkers, in early detection of CRC and AP. Material and methodsFecal samples (n = 93) were collected from Taleghani Hospital, Tehran, Iran, between 2015 and 2017, from normal controls (NC), AP cases and CRC stage I patients, who were undergoing screening for colonoscopy. Absolute quantitative real time PCR (qPCR) assays were established for the quantification of bacterial marker candidates, in all cases and control groups. In order to evaluate the diagnostic value of bacterial candidates in distinguishing CRC from a polyp, receiver operating characteristic curve (ROC) was performed. Multiple logistic regressions were used to find the best combinations of the bacterial candidates, then, combinations were analyzed based on three methods, including linear combination, multiple logistic and factor analysis models. ResultsAccording to the logistic model, combination of Fusobacterium nucleatum, Enterococcus feacalis, Streptococcus bovis, Enterotoxigenic Bacteroides fragilis (ETBF) and Porphyromonas spp. showed improved diagnostic performance, compared to each bacterium alone, as area under the receiver operating characteristic (AUROC) increases to 0.97, with 95% confidence interval. It was found that a simple linear combination was an appropriate model for discriminating AP and CRC cases, compared to the NC, with a sensitivity of 91.4% and specificity of 93.5%. ConclusionOur results indicated that based on fecal bacterial candidates, statistical simple linear combination model and ROC curve analysis, early detection of AP and CRC might be possible.

The association between fecal microbiota and different types of colorectal polyp as precursors of colorectal cancer

BackgroundColon cancer (CRC) is a heterogeneous disorder, arising from precursors-adenoma and serrated polyp. Previous studies have demonstrated the relationship between the human gut microbiota and CRC; however, its correlation to the different early precursors of CRC is not properly understood. Here, we studied the relationship between targeted gut bacteria and different colorectal polyp types, location, size and grade of dysplasia. Material and methodsIn the present case-control descriptive study, selected fecal bacteria were assessed in 118 patients, referred for standard screening colonoscopy, including 31 normal controls, 21 hyperplastic polyp (HP), 16 sessile serrated polyp (SSA), 29 tubular adenoma (TA) and 21 villous/tubuvillous polyp (VP/TVP) cases, between 2015 and 2017, by absolute quantitative real time PCR technique (q PCR) in different ethnicity of Iranian population. The panel of bacteria was including Streptococcus bovis/gallolyticus, Enterococcus faecalis, Enterotoxigenic Bacteroides fragilis (ETBF), Fusobacterium nucleatum, Porphyromonas spp., Lactobacillus spp., Roseburia spp., and Bifidobacterium spp. ResultsHigher numbers of F. nucleatum, E. feacalis, S. bovis, ETBF and Porphyromonas spp. were detected in AP cases, consisting TA and especially VP/TVP, in contrast to samples from the normal, HP and SSA groups (P < 0.001). On the contrary, lower number of Lactobacillus spp., Roseburia spp. and Bifidobacterium spp. were detected in AP, compared to the normal, HP and SSA. Surprisingly, a significant correlation was found among selected gut bacterial quantity, the size, location and grade of dysplasia of polyp cases. DiscussionThese findings suggest that gut bacteria might contribute in early stages of colorectal carcinogenesis through the development of AP, but not SSA. In fact, AP and SSA are also different in terms of molecular pathways and tendencies to present in specific colorectal location. Overall, these findings may lead to development of CRC prevention therapies, targeting early protagonist bacteria of colorectal carcinogenesis from AP.

BMP15 mRNA profile in granulosa cells from endometriosis patients undergoing in vitro fertilization

Endometriosis is a female reproductive disorder that can cause infertility by lowering oocyte quality due to a low quality of oocytes. Granulosa cell assessment is a minimally invasive procedure for determining oocyte quality. Bone morphogenetic protein 15 (BMP15) also acts as a growth factor for oocyte derivatives that regulate proliferation and granulosa cell differentiation. In endometriosis, higher BMP15 expression is assumed to induce oocyte growth. This study aimed to determine the mRNA levels of BMP15 in granulosa cells from endometriosis patients undergoing in vitro fertilization. We analyzed samples from 12 patients with endometriosis and seven normal women using real-time PCR at Yasmin Clinic, Kencana, Cipto Mangunkusumo Hospital. Granulosa cells collected during oocyte retrieval were preserved using RNAlater® QIAGEN and were evaluated by absolute quantitative real-time PCR with pre-manufactured oligonucleotides as the standard. Results showed that although BMP15 did not significantly differ between the endometriosis patients and the controls (p > 0.05), the endometriosis patients had higher BMP15 mRNA levels than the controls. Therefore, further study is warranted to gain more comprehend data.

Expression of the oxygen-sensitive transcription factor subunit HIF-1α in patients suffering from secondary Raynaud syndrome.

Anti-ischemic therapy remains a challenge due to the complexity of hypoxia response pathways. Hypoxia-inducible factor (HIF)-1 is a heterodimer transcription factor consisting of 2 subunits, HIF-1α and HIF-1β. Hypoxia-dependent activation of HIF-1α regulates cellular O2 homeostasis. Raynaud syndrome (RS), as a comorbidity of the autoimmune disease systemic sclerosis (SS), is characterized by vasospasms that limit blood flow to the limbs, resulting in hypoxia. A single-center randomized study was conducted to compare prostaglandin E1 (PgE1) therapy with a treatment combining PgE1 and an endothelin-1 blocker, bosentan. A total of 30 patients suffering from SS with RS were enrolled. We examined the regulation of HIF-1α, its target heme oxygenase-1 (HMOX-1), and the serum levels of the HIF-1α protein in a subset of patients as well as in ten healthy individuals. The expression of HIF-1α and HMOX-1 in monocytes was measured using absolute plasmid-based quantitative real-time PCR, whereas serum HIF-1α levels were measured with ELISA. Samples were taken at the time of randomization and after 24 weeks. We found that HIF-1α and HMOX-1 mRNA expression in monocytes and serum HIF-1α protein levels were significantly higher in the SS/RS patients compared to the healthy control group. Single-drug therapy significantly increased HIF-1α and HMOX-1 mRNA expression in monocytes and serum HIF-1α protein levels in the SS/RS patients compared to those at the time of randomization, whereas combining PgE1 with an endothelin-1 blocker prevented the further increases in HIF-1α and HMOX-1 expression. We propose HIF-1α and HMOX-1 as novel markers for anti-ischemic therapy in RS.

Development of an Absolute Quantitative Real-Time PCR (qPCR) for the Diagnosis of Aeromonas hydrophila Infections in Fish

Development of an Absolute Quantitative Real-Time PCR (qPCR) for the Diagnosis of Aeromonas hydrophila Infections in Fish