Real-time Polymerase Chain Reaction Quantification Research Articles

Microarray analysis reveals a potential role of LncRNAs expression in cardiac cell proliferation.

BackgroundLong non-coding RNAs (LncRNAs) have been identified to play important roles in epigenetic processes that underpin organogenesis. However, the role of LncRNAs in the regulation of transition from fetal to adult life of human heart has not been evaluated.MethodsImmunofiuorescent staining was used to determine the extent of cardiac cell proliferation. Human LncRNA microarrays were applied to define gene expression signatures of the fetal (13–17 weeks of gestation, n = 4) and adult hearts (30–40 years old, n = 4). Pathway analysis was performed to predict the function of differentially expressed mRNAs (DEM). DEM related to cell proliferation were selected to construct a lncRNA-mRNA co-expression network. Eight lncRNAs were confirmed by quantificational real-time polymerase chain reaction (n = 6).ResultsCardiac cell proliferation was significant in the fetal heart. Two thousand six hundred six lncRNAs and 3079 mRNAs were found to be differentially expressed. Cell cycle was the most enriched pathway in down-regulated genes in the adult heart. Eight lncRNAs (RP11-119 F7.5, AX747860, HBBP1, LINC00304, TPTE2P6, AC034193.5, XLOC_006934 and AL833346) were predicted to play a central role in cardiac cell proliferation.ConclusionsWe discovered a profile of lncRNAs differentially expressed between the human fetal and adult heart. Several meaningful lncRNAs involved in cardiac cell proliferation were disclosed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0139-4) contains supplementary material, which is available to authorized users.

The response of ammonia-oxidizing microorganisms to trace metals and urine in two grassland soils in New Zealand.

An incubation experiment was conducted to investigate the response of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and the nitrification rate to the contamination of Cu, Zn, and Cd in two New Zealand grassland soils. The soils spiked with different concentrations of Cu (20 and 50mgkg-1), Zn (20 and 50mgkg-1), and Cd (2 and 10mgkg-1) were incubated for 14days and then treated with 500mgkg-1 urine-N before continuing incubation for a total of 115days. Soils were sampled at intervals throughout the incubation. The nitrification rate in soils at each sampling period was determined, and the abundance of AOB and AOA was measured by real-time quantification polymerase chain reaction (qPCR) assay of the amoA gene copy numbers. The results revealed that moderate trace metal stress did not significantly affect the abundance of AOB and AOA in the two soils, probably due to the high organic matter content of the soils which would have reduced the toxic effect of the metals. Nitrification rates were much greater and the observable nitrification period was much shorter in the dairy farm (DF) soil, in which the AOB and AOA abundances were greater than those of the mixed cropping farm (MF) soil. AOB were shown to grow under high nitrogen conditions, whereas AOA were shown to grow under low N environments, with different metal concentrations. Therefore, nitrogen status rather than metal applications was the main determining factor for AOB and AOA growth in the two soils studied.

Portulacerebroside A inhibits adhesion, migration, and invasion of human leukemia HL60 cells and U937 cells through the regulation of p38/JNK signaling pathway.

Acute myeloid leukemia (AML) is a highly malignant hematopoietic tumor. This study aimed to explore the effect of portulacerebroside A (PCA) on the adhesion, migration, and invasion in human leukemia HL60 cells and U937 cells and clarify the possible mechanisms involved, which could provide potential strategies for the treatment of AML. By methyl thiazolyl tetrazolium analysis, it was found that PCA (1–10 μM) suppressed the cell viability in a time- and dose-dependent manner. A total of 1, 2, and 5 μM of PCA dramatically inhibited the adhesion, migration, and invasion of HL60 cells and U937 cells in a dose-dependent manner. Phosphorylation level of JNK and P38 protein level was measured by Western blot. After the real-time quantification polymerase chain reaction and Western blot detection of the total RNA and protein, messenger RNA, and protein expression levels of Ras homologous C (RhoC), metastasis-associated gene 1 (MTA1) and matrix metalloproteinase-2/9 (MMP-2/9) were decreased significantly in a dose-dependent manner. The phosphorylation level of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) was decreased dramatically in HL60 cells and U937 cells after PCA treatment. In conclusion, PCA significantly inhibits the adhesion, migration, and invasion of HL60 cells and U937 cells by suppressing the p38/JNK pathway and regulating the expressions of related genes.

Mild hypothermia protects rat neuronal injury after intracerebral hemorrhage via attenuating endoplasmic reticulum response induced neuron apoptosis

Background and purposeMild hypothermia has been proved to reduce global and focal cerebral ischemic injury in rodents by preventing cellular apoptosis through several pathways. However, whether hypothermia will be beneficial for intracerebral hemorrhage (ICH) and its underlying mechanisms haven’t reached a consensus. It has been implicated that endoplasmic reticulum (ER) stress plays a role in the secondary injury after ICH in rats. In this study, we aimed to investigate whether mild hypothermia would attenuate ICH induced neuronal injury via regulating ER stress. MethodsThe model of ICH was induced by injecting autologous blood (120μl) into the rat striatum. Rats were divided into sham, normothermic (NT) and hypothermic (HT) groups. HT group were subjected to mild hypothermia (33°C–35°C) for 2days starting from 6h after ICH. Neurological deficits were evaluated. The ER stress related proteins (GRP78, CHOP and p-eIF2α) and the apoptosis associated indicators (cleaved caspase3, Bcl-2 and Bim) around hematoma were assessed by western blot, qRT-PCR (quantificational real-time polymerase chain reaction), immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. ResultsNeurological deficits following ICH were reduced in HT group compared to NT group. Protein levels of GRP78, CHOP and p-eIF2α significantly increased after ICH in both NT and HT group compared to sham group, which was consistent with the trend of cleaved-caspase3 at protein level, and Bim, Bcl-2 at gene level. In comparison to NT group, GRP78, CHOP, p-eIF2α, cleaved caspase-3 and Bim all decreased, while Bcl-2 increased in HT group. Additionally, apoptotic cells detected by TUNEL staining significantly decreased in the HT group. ConclusionMild hypothermia could attenuate ICH caused neuron injury by decreasing ER response-induced neuron apoptosis.

Long Non-coding RNA HOTAIR Expression in Diffuse Large B-Cell Lymphoma: In Relation to Polycomb Repressive Complex Pathway Proteins and H3K27 Trimethylation.

BackgroundA long non-coding RNA hox transcript antisense intergenic RNA (HOTAIR) is involved in epigenetic regulation through chromatin remodeling by recruiting polycomb repressive complex 2 (PRC2) proteins (EZH2, SUZ12, and EED) that induce histone H3 trimethylation at lysine 27 (H3K27me3). Deregulation of c-MYC and interaction between c-MYC and EZH2 are well known in lymphomagenesis; however, little is known about the expression status of HOTAIR in diffuse large B-cell lymphomas (DLBCLs).MethodsThe expression status of PRC2 (EZH2, SUZ12, and EED), H3K27me3, c-MYC, and BCL2 was analyzed using immunohistochemistry (n = 231), and HOTAIR was investigated by a quantification real-time polymerase chain reaction method (n = 164) in DLBCLs.ResultsThe present study confirmed the positive correlation among PRC2 proteins, H3K27me3, and c-MYC in DLBCLs. Expression level of HOTAIR was also positively correlated to EZH2 (p < .05, respectively). Between c-MYC and HOTAIR, and between c- MYC/BCL2 co-expression and HOTAIR, however, negative correlation was observed in DLBCLs (p < .05, respectively). High level of H3K27me3 was determined as an independent prognostic marker in poor overall survival (hazard ratio, 2.0; p = .023) of DLBCL patients. High expression of HOTAIR, however, was associated with favorable overall survival (p = .004) in the univariate analysis, but the impact was not significant in the multivariate analysis. The favorable outcome of DLBCL with HOTAIR high expression levels may be related to the negative correlation with c- MYC expression or c-MYC/BCL2 co-expression.ConclusionsHOTAIR expression could be one of possible mechanisms for inducing H3K27me3 via EZH2-related PRC2 activation, and induced H3K27me3 may be strongly related to aggressive DLBCLs which show poor patient outcome.

Immune signature of urothelial cancer associated with grade, recurrence, and invasion

BackgroundUrothelial carcinoma (UC) is one of most common genitourinary malignancy and the spectrum of disease ranges from in situ lesions to muscle-invasive cancers. The non–muscle-invasive lesions have tendency to recur or progress to muscle-invasive disease. The study of the immune profile may identify immune determinants associated with high-grade, recurrence, and invasion in patients with UC. MethodsPathway-focused RT2 profiler arrays were used to screen patients with UC for dysregulation of candidate genes of Th1-Th2-Th3 and NFκB pathways, which were then validated by real-time polymerase chain reaction on tumor samples and correlated with grade, recurrence, and invasion of tumors to identify their role in predicting behavior of the tumor. The cytokines found associated with recurrence were then validated in urine of patients with UC. ResultsIFNγ, IL2, IL4, IL10, IL17, CCL7, CTLA4, and SPP1 of the cytokine pathway and TLR4, TLR3, RELA, NFκB1, and MYD88 of the NFκB pathway were found differentially expressed in patients with urothelial cancer by array and quantative real-time polymerase chain reaction. Among these, IL10 and SPP1 were found consistently up-regulated in high-grade, invasive, and recurrent cases and up-regulated IL10 and CTLA4 were found associated with a short recurrence-free survival time (P = 0.001 and P = 0.065). Urinary IL10 concentration was significantly higher in both patients with cancer and cystitis compared with healthy controls, but the difference in concentration between patients with cancer and cystitis patients was not statistically significant. However, urinary CTLA4 concentrations were found to be significantly higher in urothelial cancer patients compared with healthy controls and cystitis cases and found to be associated with poor recurrence-free survival. ConclusionThe study indicates that high urinary CTLA4 concentration raises the index of suspicion of recurrence in a known case of urothelial cancer and may be used as a surveillance marker.

Detection of Herpes Simplex Virus Infection in Patients With Ongoing Miscarriage Using Serological Tests and Real-Time Polymerase Chain Reaction

Background: Herpes simplex virus (HSV) is one of the most frequent viruses affecting females’ sexual and reproductive health. Objectives: The current study aimed to determine the HSV serostatus and viral shedding in patients with ongoing miscarriage. Methods: Two hundred and eight females were included in the study; IgM antibodies against HSV1/2 were detected in serum samples; the real-time polymerase chain reaction (PCR) quantification of viral DNA was performed on cervicovaginal samples. Positive females were tested for IgG anti-HSV-2. Results: The results indicated 12.5% IgM-positive and 2.9% real-time PCR positive samples. None of the patients was positive for the both analyses, simultaneously. Among IgM-positives cases, 16.6% were also IgG-positive; whilst in PCR-positives samples, 20% were also IgG-positive. The presence of viral DNA without detectable IgM or IgG antibodies could indicate a recent infection or a reactivation with low copy numbers. Conclusions: IgM alone is not a marker for viral shedding in genital tract. Molecular testing in conjunction of IgG test should be evaluated as an option to determine HSV status, and applied for research on HSV genital infections records. Keywords: Herpes, Diagnosis, Viral Shedding

Cloning, subcellular lacalization and expression analysis of chloroplast-targeted Obg gene in Cyathula officinalis

According to ObgC gene sequences from Cyathula officinalis genomic data, the specific primers were designed, and a full-length CoObgC cDNA (2 226 bp) was obtained by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methord. Sequence alignment showed that CoObgC gene contained a 1 818 bp open reading frame (ORF) encoding 605 amino acids. Sequence analysis predicted that molecular weight of CoObgC protein was about 66.39 kDa, the academic isoelectric point was 5.35, and the protein was stable protein. Then multiple sequence alignment was applied to construct phylogenetic tree. The real-time fluorescence quantification PCR (RT-qPCR) demonstrated that a high expression level in leaf, followed by root and flower, the low transcription was in stem. The recombinant vector pCABIA2300-CoObgC was constructed and introduced into tobacco epidermal cells by agrobacterium-mediated transformation, green fluorescence was tested and targeted to chloroplast under a laser scanning confocal microscope. These findings will be helpful to lay a foundation for studying the structure and function of CoObgC gene, and elucidating C. officinalis molecular biology experiment.

Allicin improves cardiac function by protecting against apoptosis in rat model of myocardial infarction.

To study the effects of allicin on cardiac function and underlying mechanism in rat model of myocardial infarction (MI). Ninety-four Wistar rats were randomly assigned to 6 groups (n=14-16 per group): sham control group [underwent thoracotomy without left anterior descending (LAD) occlusion and only received an injection of the same amount of citrate buffer], MI control group (subjected to LAD occlusion and only received an injection of same amount of citrate buffer), positive control group (subjected to LAD occlusion and received an injection of diltiazem hydrochloride at the dose of 1.5 mg/kg), and MI + allicin groups (subjected to LAD occlusion and received an injection of allicin at the doses of 1.2, 1.8, and 3.6 mg/kg). All of the drugs were administered intraperitoneally daily for 21 days. The infarct area was measured by myocardial staining. Hematoxylin-eosin staining was used to observe the pathological changes. Cardiac function parameters were assessed by echocardiography. The myocardial apoptotic index was estimated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. The expression of Bax and Bcl-2 were detected by quantificational real-time polymerase chain reaction and Western blot. Treatment with allicin could attenuate the myocardial infarct area (P<0.05) and relieve the changes of the myocardium. The left ventricular anterior wall diastolic and systolic thicknesses were increased in the allicin-treated groups (P<0.05), while there was no signifificant difference in the left ventricular posterior wall diastolic and systolic thickness (P>0.05). The left ventricular internal diameter in systole, ejection fraction, fractional shortening, and stroke volume were dramatically elevated in allicin-treated rats (P<0.05). Allicin dose-dependently reduced creatine kinase and lactate dehydrogenase levels (P<0.05). The myocardial apoptotic index was also markedly lowered, and Bax expression was signifificantly decreased, whereas Bcl-2 expression exhibited an opposite trend in allicin-treated rats (P<0.05). Allicin appears to exert a cardioprotective effect that may be linked to blocking Bcl-2/Bax signaling pathway-denpendent apoptosis, further improving cardiac function.

Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells.

PurposeThe purpose of this study is to examine the role of the double-stranded RNA (dsRNA) activated Toll–interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) signal pathway in triggering apoptosis in hepatocellular carcinoma (HCC) cells.Materials and MethodsFirst, siRNA targeted autophagy–related gene LC3 (pU6H1-LC3 siRNA and siLC3) and a dsRNA used as a Toll-like receptor 3 (TLR3) ligand was constructed and synthesized, respectively. Then, a human HCC cell line was transfected with dsRNA, siLC3, and cotransfected with siLC3 and dsRNA (siLC3+dsRNA), respectively. Finally, quantification real-time polymerase chain reaction, western blotting, and immunofluorescence staining were used in the HCC line (SMMC7721), and MTT assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, and transmission electron microscopy were used in an HCC xenograft model of nude mice. Human umbilical vein endothelial cell tube forming assay, color Doppler ultrasonographic flow image examination, and CD34-positive microvessel density were used in vitro and in vivo.ResultsCompared with untreated cells, the protein and mRNA expression of TLR3 and TRIF was up-regulated, in order, siLC3+dsRNA, dsRNA, and siLC3. Expression of LC3 was obviously down-regulated and the autophagosomes were significantly decreased in siLC3+dsRNA and siLC3, whereas in dsRNA (p < 0.05). LC3 and TRIF colocation was observed in HepG2 cells. Decreased cell viability, increased apoptosis, decrease in xenograft tumor volume, and angiogenesis potential were also observed in order (p < 0.05).ConclusionSuppression of intracellular autophagy resulted in decreased degradation of TRIF protein, which can promote triggering of apoptosis by the TLR3-TRIF pathway. dsRNA and siLC3 could play anticancer roles in coordination.

Characterization and genetic manipulation of primed stem cells into a functional naïve state with ESRRB.

AIMTo identify differences between primed mouse embryonic stem cells (ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state.METHODSWe have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras. Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies.RESULTSMost results show that both primed and naive cells have similar features (morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the naïve cells relative to the primed cells. Key upregulated genes in naïve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells.CONCLUSIONAlthough there are no notable phenotypic differences, there are key differences in gene expression between these naïve and primed stem cells. These differences can be overcome through overexpression.

Epidermal growth factor receptor expression in gastric tumors and its relationship with the germline polymorphisms - 216 G>T, -191 C>A, (CA) n IVS1, and R521K.

Gastric cancer (GC) is the third worldwide leading cause of cancer-related death affecting both sexes. The aberrant expression of epidermal growth factor receptor (EGFR) gene has been detected in many human epithelial malignancies and linked to advanced disease, more aggressive phenotype, and poor prognosis. To analyze the relation that the expression of EGFR in gastric tumors holds with pathological characteristics and with the germline polymorphisms -216 G>T, -191 C>A, (CA) n IVS1, and R521K. We studied 22 biopsies from gastric tumors obtained by endoscopy. EGFR expression was determined by relative quantification real-time polymerase chain reaction with the glyceraldehyde-3-phosphate dehydrogenase reference gene (as for messenger RNA [mRNA]) and by immunohistochemistry (IHC) (as for protein). EGFR germline polymorphisms were analyzed by sequencing, GeneScan, and restriction fragment length polymorphisms. EGFR mRNA expression was increased (>2-fold) in 13.6% of GC cases, decreased (<0.5-fold) in 68.2%, and normal in 18.2%; overexpression was related to well-differentiated gastric tumors, whereas underexpression was linked to moderate or poorly differentiated gastric tumors (P < 0.001). EGFR protein expression was high (IHC 2+ and 3+) in 29.4% of gastric tumors and was normal or low (score 0 to 1+) in 70.6% cases. EGFR expression, in both mRNA and protein, was not related to any EGFR polymorphism (P > 0.05). Most gastric tumors showed low EGFR expression (mRNA and protein), whereas EGFR overexpression was related to well-differentiated gastric tumors. Furthermore, germinal polymorphisms -216, -191, (CA) n IVS1, and R521K were not related to EGFR expression (mRNA or protein).

Correlation between Expression of Signal Transducer and Activator of Transcription 3 and Pituitary Adenoma Subtypes

To investigate the correlation between signal transducer and activator of transcription 3 (STAT3) expression and pituitary adenoma subtypes. The STAT3 expression profiles in different pituitary adenomas from 74 patients were determined using quantificational real-time polymerase chain reaction,Western blot,and immunohistochemistry. Expression of STAT3 was observed in all pituitary adenoma subtypes. The STAT3 expression level was highest in growth hormone adenoma when compared with other tumors including prolactin,follicle-stimulating hormone/luteinizing hormone-secreting adenoma,and adrenocorticotrophic hormone-secreting adenoma. The follicle-stimulating hormone/luteinizing hormone adenomas exhibited the lowest STAT3 expression levels. STAT3 is differentially expressed in pituitary adenoma subtypes, suggesting the cell-specific features of STAT3 regulation,although further investigations are still warranted to clarify the underlying mechanisms.

Penicillin-Binding Protein 5 Sequence Alteration and Levels of plp5 mRNA Expression in Clinical Isolates of Enterococcus faecium with Different Levels of Ampicillin Resistance.

Eighty-two nonduplicated ampicillin-resistant Enterococcus faecium (AREF) isolates from clinical infections at the Charles Nicolle Hospital of Tunisia were investigated. They were collected from January 2001 to December 2009. Genetic relationship between them was studied using pulsed-field gel electrophoresis. The amino acid sequence difference variations of the C-terminal part of penicillin-binding protein 5 (PBP5) versus levels of expressed mRNA were investigated by polymerase chain reaction (PCR), sequencing, and real-time PCR quantification of (PBP5), respectively. No β-lactamase activity was detected and none of our strains showed resistance to glycopeptides, which retain their therapeutic efficiency against enterococcal infections in our hospital. Pattern analysis of the strains revealed six main clones disseminating in different wards. Sequence data revealed the existence of 19 different plp5 alleles with a difference in 16 amino acid positions spanning from residue 414 to 632. Each allele presented at least five amino acid substitutions (His-470→Gln, Asn-496→Lys, Ala-499→Thr, Glu-525→Asp, and Glu-629→Val). No correlation between amino acid sequence polymorphism of PBP5 and levels of ampicillin resistance was detected. The levels of plp5 mRNA expression varied between strains and did not always correlate with levels of ampicillin resistance in clinical AREF.

MiR-21: A gene of dual regulation in breast cancer.

Breast cancer is characterized by an elevated capacity for tumor invasion and lymph node metastasis, but the cause remains to be determined. Recent studies suggest that microRNAs (miRNAs) can regulate the evolution of malignant behavior by regulating multiple target genes. A key oncomir in carcinogenesis is miR-21, which is consistently upregulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and searched for miR-21 targets.Total RNA from breast cancer tissue and corresponding adjacent normal tissue was extracted and used to detect miR-21 expression by quantificational real-time polymerase chain reaction (qRT-PCR), followed by analysis of the correlation between gonad hormone indices in peripheral blood and miR-21 expression in cancerous tissues from the same patients. Cell proliferation, colony formation, migration and invasion were then examined to determine the role of miR-21 in regulating breast cancer cells. Finally, western blotting was performed to determine if miR-21 regulated expression of signal transducers and activators of transcription 3 (STAT3), and assays of cell proliferation, colony formation, migration and invasion were performed to examine the role of STAT3 in regulation of breast cancer cells. We found that expression of miR-21 increased from normal through benign to cancerous breast tissues. Enhanced miR-21 expression was associated with serum levels of follicle-stimulating hormone, estradiol, β-human chorionic gonadotropin, testosterone and prolactin in patients with breast cancer. Furthermore, cell proliferation, colony formation, migration and invasion were increased after overexpression of miR-21 in breast cancer cells and reduced by miR-21 suppression. In addition, we identified a putative miR-21 binding site in the 3'-untranslated region of the STAT3 gene using an online bioinformatical tool. We found that protein expression of STAT3 was significantly downregulated when breast cancer cells were transfected with miR-21 mimics, and was significantly upregulated in breast cancer cells transfected with a miR-21 inhibitor. Finally, we found that cell proliferation, colony formation, migration and invasion were decreased by treatment with 2.5nM of Stattic, an inhibitor of STAT3 activation. Our data suggest that miR-21 expression is increased in breast cancer and plays an important role as a tumor gene by targeting STAT3, which may act as a double-response controller in breast cancer.

Different Leaching Efficiency and Microbial Community Structure Variation in Chalcopyrite Bioleaching Process Based on Different Initial Microbe Proportions

In order to investigate the effect of initial proportions of microorganisms on chalcopyrite leaching, six typical bioleaching strains were used to rebuild different co-culture systems. According to their different energy use types,Acidithiobacillus ferrooxidansDX2012 (ferrous and sulfur),Sulfobacillus thermosulfidooxidnasDX2012 (ferrous and sulfur),Leptospirillum ferriphilumDX12 (only ferrous),FerroplasmathermophilumL1 (only ferrous),Acidithiobacillus caldusDX2012 (only sulfur),Acidithiobacillus thiooxidansA01 (only sulfur) were classified to construct four groups(cells): a, 1:1:1:1:1:1; b, 10:10:1:1:1:1; c, 1:1:10:10:1:1; d, 1:1:1:1:10:10. The results showed that leachate pH of consortium a, d firstly began to decline until stabilized at 1.4. On day 9th, Eh of consortium d began to sharply rise and finally stabilized at 720 mV. Compared with consortium a, b and c, copper concentration of consortium d increased to the highest level of 6000mg/L. All the evidence suggests consortium d is the best combination which held the highest leaching rate of copper. Real-time quantification polymerase chain reaction (RT-qPCR) was used to analyze the population dynamics during all the bioleaching process. Final microbial community structure of four consortia was trended roughly the same and consortium d was first to reach final community structure on day 27th. It is concluded that bioleaching co-culture system containing a higher proportion of sulfur-oxidizing bacteria works more efficiently.

Influence of Biological and Environmental Factors on Leaching Efficiency during Chalcopyrite Bioleaching Process

In the bioleaching process, many factors in ore, water and sediments are important contributors as driving forces to improve leaching efficiency. In this study, FeSO4·7H2O and S0as energy substrates were added to an oxide-rich, low-grade copper ore to explore the influence of biological and environmental factors on leaching efficiency. Mantel test results showed that the leaching rate was significantly correlated with biological and environmental factors with r values of 0.78 and 0.63 respectively, indicating that influence of biological factors were more significant. It cannot be ignored that environmental factors were important to construct the microbial community. Real-time quantification polymerase chain reaction (RT-qPCR) showed that the dominant and minor species wereA. caldusandA. ferrooxidans. Canonical correspondence analysis (CCA) made it clear that pH and ferrous concentration were crucial for shaping microbial community. All results are of great significant to gain the optimal leaching condition on Zambia oxide-rich, low-grade copper ore.

Isolation of human serum exosome and the clinical value of exosomal miRNA detection

Objective To isolate and identify exosomes from human serum, explore the feasibility of detecting exosomal miRNA in human serum. Methods Retrospective study. Serum samples from 10 healthy individuals in January 2013 were randomly selected. Besides, from January 2013 to December 2014, serum samples from prostate cancer(PCa) patients (n=20), benign prostatic hyperplasia(BPH) patients (n=20) and healthy controls (n=20) were selected. Exosomes were isolated from these serum samples using ExoQuick, and then identified by using transmission electron microscopy, NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype. The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser. Then quantificational real-time polymerase chain reaction(qRT-PCR) was carried out to detect miRNAs in different components of human serum, and nonparametric tests were used for difference analysis. Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40-100 nm, and with maximum peak distribution of 58 nm. Moreover, they expressed HSP70 and four transmembrane protein CD63. Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA. qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome, and the expression of miRNAs in exosome pellets were higher than the whole serum (miR-21, U=16, P=0.007 2; miR-16, U=3, P<0.000 1; miR-20a, U=2, P<0.000 1; let-7a, U=13, P=0.003 2) and exosome-depleted supernatant (miR-21, U=15, P=0.006 5; miR-16, U=2, P<0.000 1; miR-20a, U=1, P<0.000 1; let-7a, U=10, P=0.002 8). miR-141, the molecular marker of prostate cancer, were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients, 20 BPH patients and 20 healthy control people. The results showed that, in three groups, exosomal miR-141 expression were all significantly higher than serum circulating miR-141 (Control group, U=66, P=0.000 3; BPH group, U=83, P=0.001 6; PCa group, U=54, P<0.000 1). In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls (3.85 fold, U=74, P=0.000 7 and 4.06 fold, U=70, P=0.000 5). Conclusion Exosome can be efficiently isolated from human serum. Compared with the whole serum, isolation of serum exosome may helpful to improve the detection of circulating miRNA.(Chin J Lab Med, 2015, 38: 557-561) Key words: Exosome; Serum; miRNA

Quantification of the APE2 gene expression level in Candida albicans clinical isolates from patients with diagnosed fungal infections.

The production of hydrolytic enzymes is considered a key virulence determinant of Candida albicans. Aminopeptidase 2 (Ape2) facilitates the penetration of C. albicans into the host tissue, by providing free amino acids to support fungal growth and proliferation. The objective of this study was to estimate the APE2 expression profile in C. albicans cells during invasion of the human epithelium. Sixty-one clinical fungal isolates and five reference strains were included in this study. The wild-type APE2 sequence was analyzed by polymerase chain reaction (PCR) amplification using genomic DNA from pathogenic isolates. Amplicons were verified in 1% agarose gel and visualized by illumination with ultraviolet (UV) light. The APE2 expression levels were analyzed with reverse transcription quantitative real-time PCR (RT-qPCR) and APE2 quantification was normalized against the reference gene in C. albicans cells grown in YEPD and during Caco-2 invasion. The APE2-specific PCR product band was found in all C. albicans and C. dubliniensis strains, but not in other common pathogenic fungi. APE2 transcript abundance was elevated in the clinical isolates growing on the Caco-2 cell line in comparison to their counterparts grown in YEPD. Our data indicate a potential role for Ape2 in the invasion of epithelial cells. APE2 expression is also strain-specific, and it is not related to isolation site or disease entity.

Tolerance and Efficacy of a Probiotic Supplement Delivered in Capsule Form

Probiotic supplementation may promote a healthy microbiome, alleviate diarrhea and constipation, and improve some metabolic parameters. Bacillus subtilis, used in fermentation of some foods, may also be tolerated and effective in a concentrated supplement form. Forty healthy male and female adults (23 ± 3.9 years of age) were randomly assigned, in a double‐blind placebo‐controlled trial, to probiotic (approximately 1.9 x 109 CFU/capsule) or placebo groups for an average of 20 days. Subjects were instructed to maintain their usual diet and activity levels over the course of the study. Fecal and fasted serum samples were collected before and after capsule consumption. Dietary and daily bowel movement (BM) frequency and consistency records were reported by subjects. Fecal DNA was subject to polymerase chain reaction (PCR) and real‐time PCR (qPCR) for microbial detection and quantification. Metabolic, lipid, and C reactive protein levels remained within normal ranges from pre‐ to post‐ capsule consumption, however serum glucose was significantly reduced in the probiotic group (91.0 to 85.9 mg/dL, α 蠄 0.05; P = 0.012). A significant increase in average number of BMs reported per day was seen in the placebo group (α 蠄 0.05; P = 0.015). No significant differences in fecal microbe content were seen in qPCR analysis, though some trends were indicated with qPCR and fecal plate count analyses. Daily consumption of B. subtilis was well tolerated and may be effective as a supplement for those with glucose intolerance and diabetes, and promote regularity of bowel movements. Limitations of the study included lack of dietary and alcohol intake control and an inadequate number of fecal subsamples for follow‐up analyses. This study was funded by Deerland Enzymes Inc.