Polymerase Chain Reaction Quantification Research Articles

Quantification of pathogens and markers of fecal contamination during storm events along popular surfing beaches in San Diego, California

Along southern California beaches, the concentrations of fecal indicator bacteria (FIB) used to quantify the potential presence of fecal contamination in coastal recreational waters have been previously documented to be higher during wet weather conditions (typically winter or spring) than those observed during summer dry weather conditions. FIB are used for management of recreational waters because measurement of the bacterial and viral pathogens that are the potential causes of illness in beachgoers exposed to stormwater can be expensive, time-consuming, and technically difficult. Here, we use droplet digital Polymerase Chain Reaction (digital PCR) and digital reverse transcriptase PCR (digital RT-PCR) assays for direct quantification of pathogenic viruses, pathogenic bacteria, and source-specific markers of fecal contamination in the stormwater discharges. We applied these assays across multiple storm events from two different watersheds that discharge to popular surfing beaches in San Diego, CA. Stormwater discharges had higher FIB concentrations as compared to proximal beaches, often by ten-fold or more during wet weather. Multiple lines of evidence indicated that the stormwater discharges contained human fecal contamination, despite the presence of separate storm sewer and sanitary sewer systems in both watersheds. Human fecal source markers (up to 100% of samples, 20-12440 HF183 copies per 100 ml) and human norovirus (up to 96% of samples, 25-495 NoV copies per 100 ml) were routinely detected in stormwater discharge samples. Potential bacterial pathogens were also detected and quantified: Campylobacter spp. (up to 100% of samples, 16-504 gene copies per 100 ml) and Salmonella (up to 25% of samples, 6-86 gene copies per 100 ml). Other viral human pathogens were also measured, but occurred at generally lower concentrations: adenovirus (detected in up to 22% of samples, 14-41 AdV copies per 100 ml); no enterovirus was detected in any stormwater discharge sample. Higher concentrations of avian source markers were noted in the stormwater discharge located immediately downstream of a large bird sanctuary along with increased Campylobacter concentrations and notably different Campylobacter species composition than the watershed that had no bird sanctuary. This study is one of the few to directly measure an array of important bacterial and viral pathogens in stormwater discharges to recreational beaches, and provides context for stormwater-based management of beaches during high risk wet-weather periods. Furthermore, the combination of culture-based and digital PCR-derived data is demonstrated to be valuable for assessing hydrographic relationships, considering delivery mechanisms, and providing foundational exposure information for risk assessment.

S100A6 promotes the proliferation and migration of cervical cancer cells via the PI3K/Akt signaling pathway.

Cervical cancer is the second most common gynecological cancer worldwide and remains one of the leading causes of cancer-associated mortality among women. S100A6 has been reported to be associated with the development of many types of cancer. The aim of the present study was to investigate the effect of S100A6 on the proliferation, apoptosis and migration of cervical cancer cells and its underlying molecular mechanisms. Quantative polymerase chain reaction (qPCR) was used to detect the basic mRNA level of S100A6 in HeLa, SiHa and CaSki cells. Western blot analysis was used to detect the protein level of S100A6, epithelial cadherin, neuronal cadherin, phosphorylated protein kinase B (p-Akt), t-Akt, p-glycogen synthase kinase 3β (GSK3β), t-GSK3β and β-catenin. Semi-qPCR was used to detect the mRNA level of Snail, Twist and Vimentin. MTT and Hoechst staining assays were used to detect the proliferation and apoptosis of cells, and wound healing and Transwell assays were used to detect the migration of cells. The results of the present study demonstrate that the levels of S100A6 were decreased in HeLa cells compared with in SiHa and CaSki cells. Overexpression of S100A6 in HeLa and CaSki cells promoted the proliferative and migratory ability, and had no significant effect on cellular apoptosis. Whereas the knockdown of S100A6 in SiHa and CaSki cells inhibited the proliferative and migratory ability, it had no significant effect on apoptosis. The overexpression of S100A6 in HeLa cells increased the levels of neuronal (N)-cadherin, vimentin, Snail and Twist. Conversely, knockdown of S100A6 in SiHa cells decreased the levels of N-cadherin, vimentin, Snail and Twist and increased the levels of epithelial (E)-cadherin. Furthermore, overexpression of S100A6 in HeLa cells activated the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, and treatment with the PI3K inhibitor LY294002 partially repressed S100A6-enhanced proliferation and migration of cervical cancer cells. These results indicate that S100A6 facilitates the malignant potential of cervical cancer cells, particularly metastatic ability and epithelial-mesenchymal transition, which is mediated by activating the PI3K/Akt signaling pathway.

Digital Polymerase Chain Reaction Quantification of SERPINA1 Predicts Prognosis in High-Grade Glioma

SERPINA1 plays an anti-inflammatory role in protecting tissues from proteolytic mechanisms. SERPINA1 is positive in gliomas by immunohistochemical analysis; however, the role of SERPINA1, including the relationship with prognosis, has been uncertain. In recent years, digital polymerase chain reaction (PCR) has provided ultra-sensitive assessment of messenger RNA expression from formalin-fixed paraffin-embedded (FFPE) tissues. In this study, we quantitatively determined the expression of SERPINA1 in high-grade gliomas (HGGs) using digital PCR, and we analyzed its relationship with prognosis. Twenty-nine FFPE surgical samples from patients with HGGs (7 of World Health Organization [WHO] grade III and 22 of WHO grade IV), and human glioblastoma cell lines, U87 and U118, were used for analysis. A qualitative assessment using immunostaining and quantitative assessment using digital PCR were performed to assess the expression of SERPINA1. The expression of SERPINA1 was demonstrated in glioma tissues and glioblastoma multiforme cell lines by immunostaining. Digital PCR analysis showed that SERPINA1 was expressed in 14.3% and 63.6% of the tissues from patients with grade III and grade IV HGG, respectively (P= 0.035). The median overall survival of 38.8 months in the low SERPINA1 expression group was longer than that of 15.3 months in the high expression group (P= 0.030). The frequency and the amount of SERPINA1 expression were higher in grade IV than in grade III HGGs. The high expression of SERPINA1 indicates a poor prognosis of HGGs.

Real-time PCR quantification of 9 periodontal pathogens in saliva samples from periodontally healthy Korean young adults.

PurposeFew studies have examined periodontal pathogens from saliva samples in periodontally healthy young adults. The purposes of this study were to determine the prevalence of periodontopathic bacteria and to quantify periodontal pathogens in saliva samples using real-time polymerase chain reaction (PCR) assays in periodontally healthy Korean young adults under 35 years of age.MethodsNine major periodontal pathogens were analyzed by real-time PCR in saliva from 94 periodontally healthy young adults. Quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter rectus, Peptostreptococcus anaerobius, and Eikenella corrodens was performed by DNA copy number measurement.ResultsF. nucleatum and E. corrodens were detected in all subjects; the numbers of positive samples were 87 (92.6%), 91 (96.8%), and 90 (95.7%) for P. gingivalis, P. anaerobius, and C. rectus, respectively. Other pathogens were also detected in periodontally healthy subjects. Analysis of DNA copy numbers revealed that the most abundant periodontal pathogen was F. nucleatum, which was significantly more prevalent than all other bacteria (P<0.001), followed by P. anaerobius, P. gingivalis, E. corrodens, C. rectus, and T. denticola. There was no significant difference in the prevalence of each bacterium between men and women. The DNA copy number of total bacteria was significantly higher in men than in women.ConclusionsMajor periodontal pathogens were prevalent in the saliva of periodontally healthy Korean young adults. Therefore, we suggest that the development of periodontal disease should not be overlooked in periodontally healthy young people, as it can arise due to periodontal pathogen imbalance and host susceptibility.

Savitzky-Golay smoothing and differentiation for polymerase chain reaction quantification.

In quantitative PCR (qPCR), replicates can minimize the impact of intra-assay variation; however, inter-assay variations must be minimized to obtain a robust quantification method. The method proposed in this study uses Savitzky-Golay smoothing and differentiation (SGSD) to identify a derivative-maximum-based cycle of quantification. It does not rely on curve modeling, as is the case with many existing techniques. PCR fluorescence data sets challenged for inter-assay variations (different thermocycler units, different reagents batches, different operators, different standard curves, and different labs) were used for the evaluation. The algorithm was compared with a four-parameter logistic model (4PLM) method, the Cy0 method, and the threshold method. The SGSD method compared favourably with all methods in terms of inter-assay variation. SGSD was statistically different from the 4PLM (P = 0.03), Cy0 (P = 0.05), and threshold (P = 0.004) methods on relative error comparison basis. For intra-assay variations, SGSD outperformed the threshold method (P = 0.005) and equalled the 4PLM and Cy0 methods (P > 0.05) on relative error basis. Our results demonstrate that the SGSD method could potentially be an alternative to sigmoid modeling based methods (4PLM and Cy0) when PCR data are challenged for inter-assay variations.

Bioaerosols concentrations in working areas in biomethanization facilities

ABSTRACTThis study sought to fill the gap in information about the type and the concentration of bioaerosols present in the air of biomethanization facilities (BF). Evaluation of bioaerosol composition and concentration was achieved in two biomethanization facilities located in Eastern Canada, during summer and winter. In order to have a thorough understanding of the studied environment, the methodology combined culture of bacteria and molds, qualitiative polymerase chain reaction (qPCR) for specific microorganisms, endotoxin quantification, and next-generation sequencing (NGS) for bacterial diversity. Results revealed that workers in biomethanization facilities are exposed to bioaerosols and pathogenic microorganisms similar to those found in composting plants. However, human exposure levels to bioaerosols are lower in BF than in composting plants. Despite these differences, use of personal protective equipment is recommended to lower the risks of health problems.Implications: Biomethanization is a new technology used in eastern Canada for waste management. In the next few years, it is expected that there will be an expansion of facilities in response of tight governmental regulations. Workers in biomethanization facilities are exposed to various amounts of bioaerosols composed of some harmful microorganisms. Therefore, monitoring this occupational exposure could be an interesting tool for improving worker’s health.

Chimerism in pediatric hematopoietic stem cell transplantation and its correlation with the clinical outcome

Hematopoietic stem cell transplantation (HSCT) is the only hope to cure many inherited and acquired hematological disorders in children. Monitoring of chimerism helps to predict the post-transplantation events, with the intention to enhance the long-term disease free survival (DFS). The study aimed to investigate the importance of early chimerism detection to predict the clinical outcome following HSCT. The study included nine recipients (six β-thalassemia and three severe aplastic anemia patients) and their 10/10 HLA identical sibling donors. Chimerism detection was performed by analysis of short tandem repeat (STR) polymerase chain reaction (PCR) for detection and quantification of the relative amounts of donor and recipient cells present on day +28. Peripheral blood (PB) was the main stem cell source for HSC transplantation. Disease free survival (DFS) was 71.4% while overall survival was 85.7% for PBSC transplants at the median follow up period of 4years. The early detection of chimerism by PCR-STR analysis for children with β-thalassemia and aplastic anemia correlated with the outcome of HSCT in 8 (88.8%) patients. Complete chimerism was associated with disease-free survival while mixed chimerism and autologous patterns were associated with poor prognosis. In conclusion, early chimerism testing is clinically important in prediction of outcome after allogeneic HSC transplantation.

Metagenomic investigation of gastrointestinal microbiome in cattle.

The gastrointestinal (GI) tract, including the rumen and the other intestinal segments of cattle, harbors a diverse, complex, and dynamic microbiome that drives feed digestion and fermentation in cattle, determining feed efficiency and output of pollutants. This microbiome also plays an important role in affecting host health. Research has been conducted for more than a century to understand the microbiome and its relationship to feed efficiency and host health. The traditional cultivation-based research elucidated some of the major metabolism, but studies using molecular biology techniques conducted from late 1980’s to the late early 2000’s greatly expanded our view of the diversity of the rumen and intestinal microbiome of cattle. Recently, metagenomics has been the primary technology to characterize the GI microbiome and its relationship with host nutrition and health. This review addresses the main methods/techniques in current use, the knowledge gained, and some of the challenges that remain. Most of the primers used in quantitative real-time polymerase chain reaction quantification and diversity analysis using metagenomics of ruminal bacteria, archaea, fungi, and protozoa were also compiled.

An improved digital polymerase chain reaction protocol to capture low-copy KRAS mutations in plasma cell-free DNA by resolving 'subsampling' issues.

Genetic alterations responsible for the initiation of cancer may serve as immediate biomarkers for early diagnosis. Plasma levels of cell‐free DNA (cfDNA) in patients with cancer are higher than those in healthy individuals; however, the major technical challenge for the widespread implementation of cfDNA genotyping as a diagnostic tool is the insufficient sensitivity and specificity of detecting early‐stage tumors that shed low amounts of cfDNA. To establish a protocol for ultrasensitive droplet digital polymerase chain reaction (ddPCR) for quantification of low‐frequency alleles within a limited cfDNA pool, two‐step multiplex ddPCR targeting eight clinically relevant mutant KRAS variants was examined. Plasma samples from patients with colorectal (n = 10) and pancreatic cancer (n = 9) were evaluated, and cfDNA from healthy volunteers (n = 50) was utilized to calculate reference intervals. Limited cfDNA yields in patients with resectable colorectal and pancreatic cancers did not meet the requirement for efficient capture and quantification of rate mutant alleles by ddPCR. Eight preamplification cycles followed by a second‐run ddPCR were sufficient to obtain approximately 5000–10 000 amplified copies per ng of cfDNA, resolving the subsampling issue. Furthermore, the signal‐to‐noise ratio for rare mutant alleles against the extensive background presented by the wild‐type allele was significantly enhanced. The cutoff limit of reference intervals for mutant KRAS was determined to be ~ 0.09% based on samples from healthy individuals. The modification introduced in the ddPCR protocol facilitated the quantification of low‐copy alleles carrying driver mutations, such as oncogenic KRAS, in localized and early‐stage cancers using small blood volumes, thus offering a minimally invasive modality for timely diagnosis.

Effect of ionizing radiation on the NKG2D ligand expression on the tumor cell SCC25 surface and the killing effect on tumor cells

Objective To investigate the effect of ionizing radiation on the expression of NKG2D ligand on the surface of oral squamous cell carcinoma cell line SCC25 and its cytotoxicity to tumor cells. Methods When SCC25 cells were cultured into logarithmic growth phase, they were randomly designed as control (without treatment) and experimental group (2 Gy ionizing radiation treatment) by drawing lots. Flow cytometry was used to detect the expressions of NKG2D ligands major histocompatibility complex class Ⅰ chain-related molecule (MIC)A, MICB, UL16 binding protein (ULBP)1 on the surface of SCC25 in the control group and the experimental group cultured for 24 h. Real-time fluorescence quantification polymerase chain reaction (RT-PCR) was used to detect the changes of NKG2D ligand mRNA expression on SCC25 cell surface after 24 h culture in the experimental group and the control group. The cells were prepared and divided into blank control group (NC), 2 Gy ionizing radiation group (R), NK1 group (target ratio was 5∶1), NK2 group (target ratio was 20∶1), NK1+ R group (target ratio was 5∶1, 2 Gy ionizing radiation), NK2+ R group (target ratio was 20∶1, 2 Gy ionizing radiation). After each group was cultured for 24 h, the killing abilities of ionizing radiation and natural killer (NK) cells to oral squamous cell carcinoma SCC25 cells were detected by CCK8. Results Flow cytometry experiment showed that, among the NKG2D ligands, the MICA fluorescence values of experimental group and control group were respectively 21.04±0.39, 22.90±0.40 (t=2.465, P=0.069), MICB fluorescence values were 27.58±0.50, 29.83±1.05 (t=1.936, P=0.125), and ULBP1 fluorescence values were 21.04±0.40, 21.78±0.50 (t=1.154, P=0.313). This indicated that after ionizing radiation on SCC25, the NKG2D ligand MICA, MICB, ULBP1 expression increased slightly, but the differences were not statistically significant. RT-PCR indicated that mRNA expressions of MICB, ULBP1 were significantly different between the control group and the experimental group (t=18.334, P=0.000; t=6.381, P=0.008). The expressions of the experimental group were respectively 6.49, 1.64 times as those of the control group. The results of CCK8 showed that, there was a significant difference in cell killing ability among NK1 group, NK2 group and NC group (F=344.600, P=0.000), suggesting that NK cells could kill tumor cells, and the higher ratio of NK cells and SCC25, the stronger killing effect. The comparison between R group and NC group showed that the difference in cell killing ability was not statistically significant (P=0.567). NK1+ R group and NK1 group were compared and the difference was not statistically significant (P=0.915). There was no significant difference between NK2+ R group and NK2 group (P=0.678). This showed that the killing effect of ionizing radiation was weak. Conclusion Ionizing radiation can increase the mRNA expression of NKG2D ligands MICB and ULBP1. This may provide a new way for tumor immunotherapy. The killing effect of ionizing radiation on cells is not obvious. It may be related to low radiation dose and only 24 h for cell culture. Key words: Radiation, ionizing; Killer cells, natural; NKG2D ligands

MicroRNA Changes in Cerebrospinal Fluid After Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) accounts for a major part of the morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). MicroRNAs (miRNAs) are pathophysiologically involved in acute cerebral ischemia. This study compared miRNA profiles in cerebrospinal fluid from neurologically healthy patients, as well as SAH patients with and without subsequent development of DCI. In a prospective case-control study of SAH patients treated with external ventricular drainage and neurologically healthy patients, miRNA profiles in cerebrospinal fluid were screened and validated using 2 different high-throughput real-time quantification polymerase chain reaction techniques. The occurrence of DCI was documented in patient charts and subsequently reviewed independently by 2 physicians. MiRNA profiles from 27 SAH patients and 10 neurologically healthy patients passed quality control. In the validation, 66 miRNAs showed a relative increase in cerebrospinal fluid from SAH patients compared with neurologically healthy patients (P<0.001); 2 (miR-21 and miR-221) showed a relative increase in SAH patients with DCI compared with those without (P<0.05) in both the screening and validation. SAH is associated with marked changes in the cerebrospinal fluid miRNA profile. These changes could be associated to the development of DCI. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01791257.

Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

Correlation between the DNA methylation and gene expression of IGFBP5 in breastcancer.

The insulin-like growth factor binding protein5 (IGFBP5) is often dysregulated in human cancers and considered neither a tumor suppressor nor an oncogene. We aim to examine the reason of the changeable gene regulation of IGFBP5 in the case of methylation in breast cancer. We used methyl-specific polymerase (MSP) chain reaction to detect CpG methylation of IGFBP5 promoter and exon-I in breast cancer and adjacent tissues. Gene expression is evaluated by quantative polymerase chain reaction (qPCR). IGFBP5 methylation was detected in 24 of 58 (41%) and 54 of 56 (96.5%) promoter and exon-I site respectively in tumor tissues. In adjacent tissues 17 of 58 (29%) and 53 of 56 (96.5%) was methylated. IGFBP5 expression was higher estrogene receptor (ER)(+) than ER(-) patients (p = 0.0549). Beside, we found a positive correlation between the expression of IGFBP5 and G2 tumor grade (p = 0.0131). However, no correlation was observed between IGFBP5 expression and age, menopause or the presence of lymph node metastasis (p > 0.05). In summary, our results showed that IGFBP5 promoter and exon-I methylation did not have any differences between tumor and adjacent tissues so that IGFBP5 methylation did not change IGFBP5 gene regulation in breast cancer. This is the first study investigating the IGFBP5 gene methylation in breast cancer.

High Blood BAALC Copy Numbers Determined By Digital Droplet PCR at Timepoint of Allogeneic Transplantation in Complete Remission Predicts Relapse in Patients with Acute Myeloid Leukemia

Allogeneic hematopoietic cell transplantation (HSCT) is a powerful consolidation option for acute myeloid leukemia (AML) patients (pts) in hematologic complete remission (CR). Disease recurrence after HSCT remains a major clinical problem & early identification of AML pts at risk of relapse is crucial to improve outcomes. High expression of the AML associated gene BAALC (Brain and acute leukemia, cytoplasmic) at diagnosis adversely impacts on outcomes in AML pts. Little is known about its prognostic capacity during disease course & as a marker of residual disease. Here we adopted digital droplet polymerase chain reaction (ddPCR) for absolute quantification of BAALC copy numbers in peripheral blood (PB) prior to HSCT in AML pts in hematologic CR.We identified 82 AML pts with PB in first (60%) or second CR (23%) or CRi (17%) up to 28 days prior to HSCT available. Median age at HSCT was 63.9 (range 50.8-76.2) years (y). All pts received non-myeloablative (NMA) conditioning (fludarabine 3x30 mg & 2 Gy total body irradiation). At diagnosis, mutation status (mut) of the NPM1, CEBPA, IDH1, IDH2,&DNMT3A gene & presence of FLT3-ITD or FLT3-TKD were assessed. In pre-HSCT PB, absolute quantification of BAALC copy numbers was performed by ddPCR & results were normalized to ABL1 copy numbers.Additionally, absolute BAALC copy numbers wereassessedin PB of healthy controls (n=7) with a median age of 62.7 (range 39.6-82.0) y.Pts were grouped according to the European LeukemiaNet (ELN) classification in 21% favorable, 23% intermediate-I, 24% intermediate-II, 23% adverse & 9% unknown. Pts & healthy control were evenly matched in age (P=1) & sex (P=1). BAALC/ABL1 copy numbers did not differ between AML pts at HSCT (median 0.03 [range 0.01-2.48]) & the healthy controls (median 0.04 [range 0.03-0.10], P=.34, Figure 1).A cut-off point of 0.14absolute BAALC/ABL1 copies was determined using the R package ’OptimalCutpoints’ & used to define pts with high (26%) & low (74%) pre-HSCT BAALC/ABL1 copy numbers. The copy number at this cut-off point was higher than the two-fold standard deviation over the median of the healthy controls (0.10 BAALC/ABL1).Pts with high & low pre-HSCT BAALC/ABL1 copy numbers did not differ significantly in pre-treatment characteristics (i.e. hemoglobin, white blood count, platelets, blasts in bone marrow or PB, ELN genetic group, FLT3-ITD, FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 mut) or remission status at HSCT (CR1 vs. CR2 vs. CRi). However, pts with high pre-HSCT BAALC/ABL1 copy numbers had a significantly higher cumulative incidence of relapse (CIR, P=.02, Figure 2a) & shorter overall survival (OS, P=.02, Figure 2b). High pre-HSCT BAALC/ABL1 copy numbers especially impacted on CIR when we restricted our analysis to pts with normal cytogenetics (P=.003). In multivariate analysis for the entire cohort, high pre-HSCT BAALC/ABL1 copy numbers retained the prognostic impact on CIR (Hazard Ratio [HR] 3.6, Confidence Interval [CI] 1.6-8.2, P=.002) after adjustment for disease status at HSCT (P=.006) & the prognostic impact on OS (HR 2.2, CI 1.1-4.3, P=.02).In conclusion, ddPCR is a feasible method for absolute quantification of BAALC copy numbers in PB, which may indicate residual disease burden in AML pts. High PB BAALC/ABL1 copy numbers (>0.14) in AML pts in hematologic CR at HSCT associated with higher CIR & shorter OS in univariate & multivariate models. AML pts with high PB BAALC/ABL1 copy numbers at HSCT should be closely monitored for relapse in the post-transplant period. In the future prospective studies will be required to validate the absolute PB BAALC/ABL1 copy number cut-off point & to evaluate whether AML pts with high BAALC/ABL1 copy numbersmight benefit from additional treatment before HSCT. [Display omitted] [Display omitted] DisclosuresPoenisch:Mundipharma: Research Funding. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

Liraglutide suppresses obesity and induces brown fat-like phenotype via Soluble Guanylyl Cyclase mediated pathway in vivo and in vitro.

Strategies for driving white adipose tissue (WAT) to acquire brown-like characteristics are a promising approach to reduce obesity. Liraglutide has been reported to active brown adipose tissue (BAT) thermogenesis and WAT browning by rapid intracerebroventricular injection in mice. In this study, we investigated the effects and possible mechanisms of liraglutide on WAT browning by chronic treatment. Here, we show that liraglutide significantly decreases body weight of mice and reduces the size of white adipocytes. By quantity polymerase chain reaction, immunoblotting analysis, cell immunofluorescence or immunocytochemical staining, we found liraglutide induced WAT browning because it up-regulated lipolytic activity, BAT, as well as mitochondrial marker genes in inguinal and peripheral renal WAT. We also confirmed liraglutide induced browning of 3T3-L1 because it enhanced expression of BAT and mitochondrial specific genes. In further, we observed that, soluble guanylyl cyclase (sGC) and protein kinase G I (PKGI) were up-regulated by liraglutide in vivo and in vitro; stimulation of sGC elevated expression of BAT markers and PKGI, which suggested that liraglutide induced WAT browning via sGC-dependent pathway. Taken together, this study expands our knowledge on the mechanism of liraglutide inducing WAT browning, and provides a theoretical support for clinical usage of liraglutide on obesity treatment.

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.

Role of phosphate groups on antiviral activity of casein phosphopeptide against feline calicivirus as a surrogate for norovirus

Current research on the gastrointestinal digestion of milk-casein strongly suggests the existence of novel bioactive peptides with antiviral activities that are attributable to their immunostimulatory effects. In the present study, we investigated the antiviral activity of casein peptides rich in phosphate groups, such as casein phosphopeptide (CPP-III). We prepared two types of CPP with different phosphorylation levels to clarify the role of the phosphate group. Further phosphorylation of CPP-III was conducted by dry heating with sodium pyrophosphate, whereas dephosphorylation was performed enzymatically using alkaline phosphatase and alkaline treatment. Feline calicivirus (FCV) strain F9, a typical norovirus surrogate, and Crandell Rees feline kidney cells were used as the target virus and host cells, respectively. Antiviral activity was determined based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and quantitative polymerase chain reaction quantification of antiviral cytokine mRNA expression. Higher cell viability was observed in the host cells treated with phosphorylated CPP-III, and a significant up-regulation of type 1 interferon expression was induced compared to that treated with native CPP-III. However, dephosphorylation of CPP-III resulted in a decrease in the anti-FCV effect. The CPP effect was enhanced by the introduction of additional phosphates and conversely weakened by their elimination. Therefore, CPP-III phosphorylation represents an emerging approach for the production of food-grade antiviral agents. © 2016 Society of Chemical Industry.

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

Recent Development of Droplet Microfluidics in Digital Polymerase Chain Reaction

Digital polymerase chain reaction (PCR) has been experiencing a rapid development during the past few years. Compared with the traditional qPCR, the accuracy of digital PCR for the absolute quantification of target gene has been significantly improved with the same primer and probe as qPCR. The development of digital PCR is directly related to the development of microfluidics. The integrated fluid circuit, which has a complicated fabrication process with high cost, is an early integration of the microfluidics and digital PCR. Recently, researchers are trying to apply the droplet microfluidics in digital PCR. A droplet microfluidic chip is able to generate millions of droplets within a short time. Each of these droplets containing no more than one target gene is a reaction chamber during the amplification process. After amplification, each droplet is tested to obtain the absolute quantification of the target gene. This paper reviews the recent progress of droplet digital PCR and the application of droplet digital PCR in biological, medical and environmental area.