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Streptococcus suis is an important porcine bacterial pathogen and a zoonotic agent responsible for sudden death, septic shock and meningitis, of which serotype 2 is the most widespread, with serotype 14 also causing infections in humans in South-East Asia. Knowledge of its pathogenesis and virulence are almost exclusively based on these two serotypes. Though serotype 9 is responsible for the greatest number of porcine cases in Spain, the Netherlands and Germany, very little information is currently available regarding this serotype. Of the different virulence factors, the capsular polysaccharide (CPS) is required for S. suis virulence as it promotes resistance to phagocytosis and killing and masks surface components responsible for host cell activation. However, these roles have been described for serotypes 2 and 14, whose CPSs are structurally and compositionally similar, both containing sialic acid. Consequently, we evaluated herein the interactions of serotype 9 with host cells and the role of its CPS, which greatly differs from those of serotypes 2 and 14. Results demonstrated that serotype 9 adhesion to but not invasion of respiratory epithelial cells was greater than that of serotypes 2 and 14. Furthermore serotype 9 was more internalized by macrophages but equally resistant to whole blood killing. Though recognition of serotypes 2, 9 and 14 by DCs required MyD88-dependent signaling, in vitro pro-inflammatory mediator production induced by serotype 9 was much lower. In vivo, however, serotype 9 causes an exacerbated inflammatory response, which combined with persistent bacterial presence, is probably responsible for host death during the systemic infection. Though presence of the serotype 9 CPS masks surface components less efficiently than those of serotypes 2 and 14, the serotype 9 CPS remains critical for virulence as it is required for survival in blood and development of clinical disease, and this regardless of its unique composition and structure. [ABSTRACT FROM AUTHOR]

Urine is an acceptable, non-invasive sample for investigating the human urogenital microbiota and for the diagnosis of sexually transmitted infections. However, low quantities of bacterial DNA and PCR inhibitors in urine may prevent efficient PCR amplification for molecular detection of bacteria. Furthermore, cold temperatures used to preserve DNA and bacteria in urine can promote precipitation of crystals that interfere with DNA extraction. Saline, Dulbecco’s Phosphate Buffered Saline, or Tris-EDTA buffer were added to urine from adult men to determine if crystal precipitation could be reversed without heating samples beyond ambient temperature. Total bacterial DNA concentrations and PCR inhibition were measured using quantitative PCR assays to compare DNA yields with and without buffer addition. Dissolution of crystals with Tris-EDTA prior to urine centrifugation was most effective in increasing bacterial DNA recovery and reducing PCR inhibition. DNA recovery using Tris-EDTA was further tested by spiking urine with DNA from bacterial isolates and median concentrations of Lactobacillus jensenii and Escherichia coli 16S rRNA gene copies were found to be higher in urine processed with Tris-EDTA. Maximizing bacterial DNA yield from urine may facilitate more accurate assessment of bacterial populations and increase detection of specific bacteria in the genital tract. [ABSTRACT FROM AUTHOR]

Physicochemical properties of water molecules as the main compositions of the freezing media can be affected by the electromagnetic fled. The purpose of this study was to apply extremely low repetition rate electromagnetic fields (ELEFs) to change the molecular network of water molecules existing in freezing media used for human sperm cryopreservation. First, different time periods and pulsed electromagnetic fields were used to evaluate the physiochemical properties of water. The lowest rate of cluster size, surface tension, viscosity, and density was observed for water samples exposed to 1000 Hz ELEF for 60 min (P < 0.05) that could be results in small ice crystal formation. Therefore, this treatment was selected for further evaluations in human sperm freezing because there was minimal probability of amorphous ice crystallization in this group. To assess fertilizing potential, human semen samples were subjected to ELEF (1000 Hz) water-made freezing medium and cryopreserved. The highest percentage of total motility, progressive motility, viability, membrane integrity, mitochondrial membrane potential, DNA integrity, and TAC were obtained in frozen ELEF as compared to other groups. The percentage of viable spermatozoa (Annexin V-/PI-) in frozen ELEF was significantly higher than in frozen control. The level of ROS was significantly lower in frozen ELEF when compared to frozen control. It can be concluded that the modification of physicochemical properties of water existing in cryopreservation media by ELEF is a suitable strategy to improve the outcome of cryopreservation. [ABSTRACT FROM AUTHOR]

Background: Although evidence of a disseminated intravascular coagulation (DIC)-like reaction has been identified in the lung parenchyma of patients with acute exacerbation of idiopathic pulmonary fibrosis (IPF), an association between DIC and IPF outcome has not been elucidated. Therefore, we retrospectively investigated the association between the Japanese Association for Acute Medicine (JAAM)-DIC score and mortality in patients with acute exacerbation of fibrosing idiopathic interstitial pneumonia (AE-fIIP). Methods: Between January 2008 and December 2016, consecutive patients with chronic fIIP who were admitted for the first time for AE-fIIP were recruited into the study. Associations between clinical data and JAAM-DIC score at the time of admission and mortality were examined. Results: During the study period, a total of 91 patients with fIIP (73.0±8.4 y.o.) were hospitalized for AE-fIIP for the first time. The 30-day and hospital mortality were 8.7% and 17.5%, respectively. A multivariate analysis showed that the JAAM-DIC score on admission was an independent predictor of 30-day mortality (odds ratio [OR] 2.57, 95% confidential interval [CI] 1.50–4.40, P = 0.0006). The APACHE II score (OR 1.29, 95% CI 1.01–1.63, P = 0.03) and the JAAM-DIC score (OR 3.47, 95% CI 1.73–6.94, P = 0.0004) were independent predictors of hospital mortality. Conclusions: The JAAM-DIC scoring system can predict survival in patients with AE-fIIP. The role of DIC in the pathogenesis of AE-fIIP merits further investigation. [ABSTRACT FROM AUTHOR]

Klotho is a soluble or membrane-bound anti-aging protein, whose protective actions are important for a prudential function of many organs. Because Klotho and cerebral small vessel disease (SVD) are associated with ageing process and endothelial dysfunction, it is possible that Klotho has an association with cerebral SVD. We aimed to investigate the association of plasma Klotho concentration with the presence, burden and progression of cerebral SVD. We prospectively enrolled 262 patients with first-ever acute cerebral infarction, performed brain MRI and collected their blood samples within 24 hours of admission. An enzyme-linked immunosorbent assay was used for evaluating plasma Klotho concentration. We estimated the total SVD score of each patient after determining the presence and burden of high-grade white matter hyperintensities (HWMHs), cerebral microbleeds (CMBs), high-grade perivascular spaces (HPVSs) and asymptomatic lacunar infarctions (ALIs). Univariate and multivariate analyses were conducted to investigate association of Klotho with cerebral SVD and the total SVD score. Of the 262 patients, 152 (58.0%) were men. The mean age of these patients was 64.7 years. The mean ± standard deviation of plasma Klotho concentration was 329.8 ± 194.1 pg/mL. In multivariate analysis, plasma Klotho concentration was negatively associated with the presence of HWMHs [Odds ratio (OR): 0.13, p = 0.047], HPVSs (OR: 0.22, p = 0.024) and ALIs (OR: 0.53, p = 0.021) but not associated with the presence of CMBs (OR: 0.39, p = 0.404). Plasma Klotho concentration was also negatively related to the total SVD score (unstandardized coefficients beta: −0.895, standard error = 0.317, p = 0.005, R2 = 0.239). Furthermore, plasma Klotho concentration was negatively related to the presence (OR: 0.75, 95% CI: 0.59–0.96, p = 0.025) and severity of cerebral SVD progression (OR: 0.72, 95% CI: 0.56–0.92, p = 0.009). In conclusion, plasma Klotho concentration was negatively associated with the presence, burden and progression of cerebral SVD. [ABSTRACT FROM AUTHOR]

Dipeptidyl amino-peptidase 3 (DPP3) is an aminopeptidase involved in peptide degradation, including hormone peptides as angiotensin II and enkephalins. DPP3 plasma activity increases in septic patients and correlates with mortality risk. However, the exact physiological role of DPP3 remains unclear and animal studies are necessary to reveal the function of DPP3 in vivo. To this demand, we developed a two-step purification procedure for isolation of native human DPP3 from blood cell lysate (BCL) that is suitable for in vivo applications. With the use of monoclonal antibodies coupled to beads in combination with an ion-exchange chromatography, we recovered 68% of human DPP3 activity from BCL with a purity of ≥ 95%. Purified human DPP3 was assayed for activity and protein concentration using recently published DPP3-activity- and immunoassays. Additionally, protein stability and storage in relevant buffers were tested. Our results provide a promising strategy for fast and efficient isolation of human DPP3. The purified human DPP3 represents the native state of DPP3, suitable for future in vivo applications to investigate the physiological role of DPP3 and its involvement in pathophysiological conditions. [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation patterns are common in cancers and environmental pollutant exposed subjects. Up to date, few studies have examined the aberrant DNA methylation patterns in benzene exposed workers. We recruited 141 benzene-exposed workers, including 83 benzene-exposed workers from a shoe factory in Wenzhou and 58 workers from a painting workshop in Wuhu, 35 workers in Wuhu were followed from 2009 to 2013, and 48 indoor workers as controls from Wenzhou. We used high-resolution melting (HRM) to quantitate human samples of DNA methylation in long interspersed nuclear element-1 (LINE-1), (6)-methylguanine-DNA methyltransferase (MGMT), and DNA mismatch repair gene human mutator L homologue 1 (hMLH1). AML-5 cells were treated with benzoquinone (BQ) and hydroquinone (HQ), and the promoter methylation of MGMT and hMLH1 was detected using the bisulfite sequencing PCR method. The degree of LINE-1 methylation in benzene-exposed workers was significantly lower than that of the controls (p<0.001), and the degree of MGMT (p<0.001) and hMLH1 (p = 0.01) methylation was significantly higher than that of the controls. The in vitro study validated the aberrant hypermethylation of hMLH1 after treatment with BQ. Among the cohort workers who were followed from 2009 to 2013, the LINE1 methylation elevated in 2013 than 2009 (p = 0.004), and premotor methylation in hMLH1 reduced in 2013 than 2009 (p = 0.045) with the reduction of the benzene exposure. This study provides evidence that benzene exposure can induce LINE-1 hypomethylation and DNA repair gene hypermethylation. [ABSTRACT FROM AUTHOR]

Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein–chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors. [ABSTRACT FROM AUTHOR]

IgA nephropathy, a common chronic kidney disease, has various possible outcomes. Therefore, the identification of novel prognostic biomarkers is needed. To this purpose, we used gas chromatography mass spectrometry to search for metabolites capable of predicting the phenotypes of IgA nephropathy in hyper IgA (HIGA) mice, an established model mice for IgA nephropathy. We measured the plasma metabolite levels in 12- and 22-week-old mice, prior to the manifestation of IgA nephropathy phenotypes, and statistically investigated the associations between these metabolites and the phenotypes of IgA nephropathy, such as the urine protein levels and histological phenotypes of the kidney at 32 weeks. We observed that in plasma samples collected from 12- and 22-week-old HIGA mice, the urinary protein levels were significantly associated with 8 and 10 metabolites, the glomerular cellular component levels were significantly associated with 8 and 7 metabolites, and the mesangial substrate levels were significantly associated with 8 and 8 metabolites, respectively. Among the candidate metabolites associated with the phenotypes of IgA nephropathy, coniferyl alcohol levels were significantly higher in HIGA mice at all of the 12, 22, and 32 weeks of age. Since this study was an observational study, we could not elucidate the underlying mechanisms; however, we were able to identify new candidate metabolites, such as coniferyl alcohol, as being potentially involved in the pathogenesis of IgA nephropathy. These results might help to develop novel laboratory tests and therapeutic reagents for IgA nephropathy in the future. [ABSTRACT FROM AUTHOR]

Background: The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature. Objectives: Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. Materials and methods: Thawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 μM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor). Results: Rosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (p<0.01), both of which were prevented by incubation in the presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (P<0.01). Conclusion: We provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology. [ABSTRACT FROM AUTHOR]

Background: Prostate cancer (PC) is a multifocal disease. DNA methylation alterations are not restricted to the immediate peritumor environment, but spatially widespread in the adjacent and distant histologically normal prostate tissues. In the current study, we utilized high-throughput methylation arrays to identify epigenetic changes in the urine from men with and without cancer. Design, setting, and participants: DNA urine samples were enriched for methylated fragments using MBD methyl-binding antibodies and applied to high density CytoScanHD arrays. Significant loci were validated using quantitative pyrosequencing and binary logistic regression modeling applied to urine sample analyses in a training (n = 83) and validation approach (n = 84). Methylation alterations in prostate tissues using pyrosequencing at the PLA2G16 locus were examined in 38 histologically normal specimens from men with (TA, n = 26) and without (NTA, n = 12) cancer and correlated to gene expression. Results: Methylation microarrays identified 3,986 loci showing significantly altered methylation in the urine samples from patients with PC compared to those without (TA vs NTA; p<0.01). These loci were then compared against subjects with their prostates removed to exclude non-prostate cell markers yielding 196 significant regions. Multiple CpGs adjacent to PLA2G16 CpG island showed increased methylation in TA compared to NTA (p<0.01) in a large validation study of urine samples. The predictive accuracy of PLA2G16 methylation at CG2 showed the highest predictive value at 0.8 (odds ratio, 1.37; 95% confidence interval, 1.16–1.62; p<0.001). Using a probability cutoff of 0.065, the sensitivity and specificity of the multivariate model was 92% and 35%. When histologically normal prostate tissues/biopsies from patients with PC (TA) were compared to subjects without cancer, significant hypermethylation of PLA2G16 was noted (odds ratio, 1.35; 95% confidence interval, 1.07–1.71; p = 0.01). Conclusion: PLA2G16 methylation defines an extensive field defect in histologically normal prostate tissue associated with PC. PLA2G16 methylation in urine and prostate tissues can detect the presence of PC. [ABSTRACT FROM AUTHOR]

Semen analysis is one of the standard diagnostic tools currently used to assess male infertility and reproductive toxicity. However, semen analysis has a limited ability to separate fertile from infertile populations. Additional methods to detect impaired fertility are needed. The purpose of the present study was to evaluate how spermatozoal RNA content varies with sociodemographic and behavior/lifestyle factors, and to determine if spermatozoal large and small RNAs discriminate normal from abnormal spermatozoa. Semen specimens were collected from 133 men aged between 18 to 55 years undergoing semen analysis as part of couple infertility evaluation while 10 proven fertile donors were recruited as control group. Semen samples were classified as normal or abnormal according to World Health Organization (WHO) 2010 criteria. Sperm RNAs were extracted after somatic cells were lysed, and the association of large or small RNA content with semen quality and sociodemographic and behavioral/lifestyle factors was evaluated using a generalized additive model and one-way ANOVA. Inverse relationship was observed between large RNA content and sperm parameters such as sperm count, density and motility. Large RNA content per sperm was significantly increased in semen samples showing abnormal number of round cells. Furthermore, sperm motility was inversely associated with spermatozoal small RNA contents. Grouping donors by the number of semen abnormalities, we observed significant increased spermatozoal large and small RNA content in men with more than two semen abnormalities. Alcohol consumption was strongly associated with increased large RNA per sperm concentration after adjustment for age and BMI. Our study demonstrates a strong relationship between spermatozoal large RNA content and poor semen characteristics that may lead to a role in the assessment of male fertility, and may be used as an endpoint for reproductive toxicology risk assessment. [ABSTRACT FROM AUTHOR]

Introduction: Atherosclerotic plaque formation is an inflammatory process that involves the recruitment of neutrophil granulocytes and the generation of reactive oxygen species (ROS). ROS formation by myeloperoxidase, a key enzyme in H2O2 degradation, can be modulated by addition of sodium thiocyanate (NaSCN). However, the therapeutic use of NaSCN to counteract atherogenesis has been controversial, because MPO oxidizes NaSCN to hypothiocyanous acid, which is a reactive oxygen species itself. Therefore, this study aimed to investigate the effect of NaSCN treatment on atherogenesis in vivo. Methods: Apolipoprotein E knockout (ApoE−/−) mice on western-diet were treated with NaSCN for 8 weeks. Blood levels of total cholesterol, IL-10, and IL-6 were measured. Aortic roots from these mice were analyzed histologically to quantify plaque formation, monocyte, and neutrophil granulocyte infiltration. Oxidative damage was evaluated via an L-012 chemiluminescence assay and staining for chlorotyrosine in the aortic walls. Endothelial function was assessed by use of endothelium-dependent vasodilation in isolated aortic rings. Neointima formation was evaluated in wild-type mice following wire injury of the carotid artery. Results: NaSCN treatment of ApoE-/- mice lead to a reduction of atherosclerotic plaque size in the aortic roots but had no effect on monocyte or granulocyte infiltration. Serum levels of the pro-inflammatory cytokine IL-6 decreased whereas anti-inflammatory IL-10 increased upon NaSCN treatment. In our experiments, we found oxidative damage to be reduced and the endothelial function to be improved in the NaSCN-treated group. Additionally, NaSCN inhibited neointima formation. Conclusion: NaSCN has beneficial effects on various stages of atherosclerotic plaque development in mice. [ABSTRACT FROM AUTHOR]

Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm. [ABSTRACT FROM AUTHOR]

Non-Hodgkin lymphomas are among the most common types of tumors in dogs, and they are currently accepted as comparative models of the disease in humans. Aberrant patterns of DNA methylation seem to play a key role in the development of hematopoietic neoplasms in humans, constitute a special mechanism of transcriptional control, and may be influenced by genetic and environmental factors. Blood leukocyte DNA global methylation has been poorly investigated in dogs. The aim of this study is to examine whether peripheral blood global DNA methylation is associated with canine multicentric lymphomas. Peripheral venous blood samples from ten healthy dogs and nine dogs bearing multicentric lymphomas were collected, and the buffy coat was separated. Global DNA methylation was analyzed by High Performance Liquid Chromatography (HPLC) and immunocytochemistry (ICC). In both analyses, leukocytes from dogs with lymphoma presented lower global DNA methylation than in healthy dogs (HPLC: p = 0.027/ 5MeCyt immunoreactivity scores: p = 0.015). Moderate correlation was observed between the results obtained by HPLC and ICC (correlation coefficient = 0.50). For the identification of differently methylated genes between both groups, the Infinium Human Methylation (HM) EPIC BeadChip (850K) was used. Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 8433 regions, and through the homologous and orthologous similarities 525 differently methylated genes were identified between the two groups. This study is pioneer in suggesting that dogs bearing non-Hodgkin lymphoma presented DNA global hypomethylation of circulating leukocytes compared with healthy dogs. Although canine samples were used in an assay developed specifically for human DNA, it was possible to identify differently methylated genes and our results reiterate the importance of the use of peripheral blood leukocytes in cancer research and possible new biomarkers targets. [ABSTRACT FROM AUTHOR]

Objective: Passive heating (PH) has begun to gain research attention as an alternative therapy for cardio-metabolic diseases. Whether PH improves glycaemic control in diabetic and non-diabetic individuals is unknown. This study aims to review and conduct a meta-analysis of published literature relating to PH and glycaemic control. Methods: Electronic data sources, PubMed, Embase and Web of Science from inception to July 2018 were searched for randomised controlled trials (RCT) studying the effect of PH on glycaemic control in diabetic or non-diabetic individuals. To measure the treatment effect, standardised mean differences (SMD) with 95% confidence intervals (CI) were calculated. Results: Fourteen articles were included in the meta-analysis. Following a glucose load, glucose concentration was greater during PH in non-diabetic (SMD 0.75, 95% CI 1.02 to 0.48, P < 0.001) and diabetic individuals (SMD 0.27, 95% CI 0.52 to 0.02, P = 0.030). In non-diabetic individuals, glycaemic control did not differ between PH and control only (SMD 0.11, 95% CI 0.44 to -0.22, P > 0.050) and a glucose challenge given within 24 hours post-heating (SMD 0.30, 95% CI 0.62 to -0.02, P > 0.050). Conclusion: PH preceded by a glucose load results in acute glucose intolerance in non-diabetic and diabetic individuals. However, heating a non-diabetic individual without a glucose load appears not to affect glycaemic control. Likewise, a glucose challenge given within 24 hours of a single-bout of heating does not affect glucose tolerance in non-diabetic individuals. Despite the promise PH may hold, no short-term benefit to glucose tolerance is observed in non-diabetic individuals. More research is needed to elucidate whether this alternative therapy benefits diabetic individuals. [ABSTRACT FROM AUTHOR]

While carbon monoxide (CO) is considered toxic, low levels of endogenously produced CO are protective against cellular injury induced by oxidative stress. Carboxyhaemoglobin (COHb) levels have been associated with outcomes in critically ill adults. We aimed to describe the distribution of carboxyhaemoglobin in critically ill children and the relationship of these levels with clinical outcomes. This retrospective observational study was conducted at a large tertiary paediatric intensive care unit (PICU). We included all children admitted to the PICU over a two-year period who underwent arterial blood gas analysis. We measured the following: (i) Population and age-related differences in COHb distribution; (ii) Change in COHb over the first week of admission using a multi-level linear regression analysis; (iii) Uni- and multivariable relationships between COHb and length of ventilation and PICU survival. Arterial COHb levels were available for 559/2029 admissions. The median COHb level was 1.20% (IQR 1.00–1.60%). Younger children had significantly higher COHb levels (p-value <2 x 10−16). Maximum Carboxyhaemoglobin was associated with survival 1.67 (95% CI: 1.01–2.57; p-value = 0.02) and length of ventilation (OR 5.20, 95% CI: 3.07–7.30; p-value = 1.8 x 10−6) following multi-variable analysis. First measured and minimum COHb values were weakly associated with length of ventilation, but not survival. In conclusion, children have increased COHb levels in critical illness, which are greater in younger children. Higher COHb levels are associated with longer length of ventilation and death in PICU. This may reflect increased oxidative stress in these children. [ABSTRACT FROM AUTHOR]

β-alanine is the rate-limiting point for the endogenous synthesis of carnosine in skeletal muscle. Carnosine has a wide range of implications for health, normal function and exercise performance. Whilst the physiological relevance of carnosine to different tissues remains enigmatic, β-alanine administration is a useful strategy to investigate the physiological roles of carnosine in humans. Intravenous administration of β-alanine is an interesting approach to study carnosine metabolism. However, sterilisation is mandatory due to the nature of the administration route. We evaluated whether sterilising doses of gamma radiation damages the molecular structure and leads to the loss of functional characteristics of β-alanine. Pure β-alanine was sterilised by gamma radiation in sealed glass vials using a 60Co multipurpose irradiator at a dose rate of 8.5 kGy.hour-1 totalising 10, 20, 25 30 and 40 kGy. The molecular integrity was assessed by X-ray Diffraction and changes in content were determined by High Performance Liquid Chromatography (UV-HPLC) and Triple Quadrupole Mass Spectrometer (HPLC/MS-MS). Sterility assurance was evaluated by inoculation assay. To examine whether functional properties were preserved, β-alanine was infused in one participant, who rated the level of paraesthesia on the skin using a 0–3 scale. Urinary β-alanine was quantified before and 24-h following β-alanine infusion using HPLC-ESI+-MS/MS. Irradiation resulted in no change in the crystal structure of β-alanine, no degradation, and no new peaks were identified in the dose range assayed. The inoculation assay showed the absence of viable microorganisms in all β-alanine samples, including those that did not undergo irradiation. Intravenous infusion of β-alanine resulted in paraesthesia and it detected in the urine as per normal. We conclude that gamma radiation is a suitable technique for the sterilisation of β-alanine. It does not lead to degradation, damage to the β-alanine structure, content or loss of function within the evaluated irradiation conditions. [ABSTRACT FROM AUTHOR]

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat (‘good freezer’) and Black-Red Andaluza (‘bad freezer’) showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages. [ABSTRACT FROM AUTHOR]

Measuring oxidative stress has become increasingly valuable in ecological studies, especially when different markers are measured on the same individual. However, many of the current methods lack sensitivity for analysis of low blood volume samples, which represent a challenge for longitudinal field studies of small organisms. Small blood volumes can usually only be analysed by using a single assay, therefore providing limited information on individual’s oxidative profile. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and modified methods presented in the literature to improve analytical selectivity and sensitivity required for small blood volumes. Specifically, we proposed a modified malondialdehyde (MDA) analysis protocol by HPLC and also optimized both the uric acid independent ferric reducing antioxidant power (FRAP) and hypochlorous acid shock capacity (HASC) assays. Development of the three modified methods was achieved with a sensitivity and repeatability that meets standards of field ecology while allowing measurement of all three assays in duplicate using less than 60 μL of plasma. Availability of these tests using small blood volumes will provide ecologists with a more comprehensive portrait of an individual’s oxidative profile and a better understanding of its determinants and interactions with the environment. [ABSTRACT FROM AUTHOR]

Epigenome-wide association studies seek to identify DNA methylation sites associated with clinical outcomes. Difference in observed methylation between specific cell-subtypes is often of interest; however, available samples often comprise a mixture of cells. To date, cell-subtype estimates have been obtained from mixed-cell DNA data using linear regression models, but the accuracy of such estimates has not been critically assessed. We evaluated linear regression performance for cell-subtype specific methylation estimation using a 450K methylation array dataset of both mixed-cell and cell-subtype sorted samples from six healthy males. CpGs associated with each cell-subtype were first identified using t-tests between groups of cell-subtype sorted samples. Subsequent reduced panels of reliably accurate CpGs were identified from mixed-cell samples using an accuracy heuristic (D). Performance was assessed by comparing cell-subtype specific estimates from mixed-cells with corresponding cell-sorted mean using the mean absolute error (MAE) and the Coefficient of Determination (R2). At the cell-subtype level, methylation levels at 3272 CpGs could be estimated to within a MAE of 5% of the expected value. The cell-subtypes with the highest accuracy were CD56+ NK (R2 = 0.56) and CD8+T (R2 = 0.48), where 23% of sites were accurately estimated. Hierarchical clustering and pathways enrichment analysis confirmed the biological relevance of the panels. Our results suggest that linear regression for cell-subtype specific methylation estimation is accurate only for some cell-subtypes at a small fraction of cell-associated sites but may be applicable to EWASs of disease traits with a blood-based pathology. Although sample size was a limitation in this study, we suggest that alternative statistical methods will provide the greatest performance improvements. [ABSTRACT FROM AUTHOR]

Genome-wide DNA methylation studies are becoming increasingly important in unraveling the epigenetic basis of cell biology, aging and human conditions. The aim of the present study was to explore whether different methods for extracting DNA from whole blood can affect DNA methylation outcome, potentially confounding DNA methylation studies. DNA was isolated from healthy blood donors (n = 10) using three different extraction methods (i.e. two automatic extractions methods based on magnetic beads or isopropanol precipitation, and manual organic extraction). DNA methylation was analyzed using the Infinium HumanMethylation450 Bead Chip (Infinium 450K) (n = 30 samples in total), which is a frequently used method in genome-wide DNA methylation analyses. Overall, the different extraction methods did not have a significant impact on the global DNA methylation patterns. However, DNA methylation differences between organic extraction and each of the automated methods were in general larger than differences between the two automated extraction methods. No CpG sites or regions reached genome-wide significance when testing for differential methylation between extraction methods. Although this study is based on a small sample, these results suggest that extraction method is unlikely to confound Infinium 450K methylation analysis in whole blood. [ABSTRACT FROM AUTHOR]

Species are shifting their ranges due to climate change, many moving to cooler and higher locations. However, with elevation increase comes oxygen decline, potentially limiting a species’ ability to track its environment depending on what mechanisms it has available to compensate for hypoxic stress. Pikas (Family Ochotonidae), cold-specialist small mammal species, are already undergoing elevational range shifts. We collected RNA samples from one population of Ochotona roylei in the western Himalaya at three sites– 3,600, 4,000, and 5,000 meters–and found no evidence of significant population genetic structure nor positive selection among sites. However, out of over 10,000 expressed transcripts, 26 were significantly upregulated at the 5,000 m site and were significantly enriched for pathways consistent with physiological compensation for limited oxygen. These results suggest that differences in gene expression may play a key role in enabling hypoxia tolerance on this local scale, indicating elevational flexibility that may facilitate successful range shifts in response to climate change. [ABSTRACT FROM AUTHOR]

This study was undertaken to evaluate a novel method for stabilizing and preserving the original proportion of cell-free fetal DNA (cffDNA) in maternal blood for extended periods of time without using crosslinking agents, such as formaldehyde, which compromise DNA integrity and extraction efficiency. Blood was drawn from pregnant donors into K3EDTA and Blood Exo DNA ProTeck® (ProTeck) tubes. Blood drawn into both tubes were aliquoted and stored at three different temperatures. At indicated times sample aliquots were processed for cell-free DNA (cfDNA) extraction. Plasma cfDNA and cffDNA quantified by droplet digital PCR (ddPCR) assay which amplify RASSF1A gene promoter region. ProTeck reagent is formaldehyde free and inhibits blood cell metabolism in blood samples during storage. Cell-free DNA concentration increased over time in blood plasma stored in K3EDTA tubes at 4, 22 and 30°C. Blood stored in ProTeck tubes, cfDNA concentration was stable at 4, 22 and 30°C for 21, 28 and 7 days, respectively. In K3EDTA tubes cffDNA proportion decreases steadily over time whereas in ProTeck tubes cffDNA proportion remained stable. This novel technology stabilizes cffDNA proportion in maternal blood samples at 4, 22 and 30°C for 21, 28 and 7 days, respectively. [ABSTRACT FROM AUTHOR]

Most patients with chronic lower back pain (CLBP) exhibit degenerative disc disease. Disc specimens obtained during initial therapeutic discectomies are often infected/colonized with Propionibacterium acnes, a Gram-positive commensal of the human skin. Although pain associated with infection is typically ascribed to the body’s inflammatory response, the Gram-positive bacterium Staphylococcus aureus was recently observed to directly activate nociceptors by secreting pore-forming α-hemolysins that disrupt neuronal cell membranes. The hemolytic activity of P. acnes in cultured disc specimens obtained during routine therapeutic discectomies was assessed through incubation on sheep-blood agar. The β-hemolysis pattern displayed by P. acnes on sheep-blood agar was variable and phylogroup-dependent. Their molecular phylogroups were correlated with their hemolytic patterns. Our findings raise the possibility that pore-forming proteins contribute to the pathogenesis and/or symptomology of chronic P. acnes disc infections and CLBP, at least in a subset of cases. [ABSTRACT FROM AUTHOR]

Glucocorticoid (GC) refractory relapses in patients with multiple sclerosis (MS) or clinically isolated syndrome (CIS), who are in potential need of treatment escalation, are a key challenge in routine clinical practice. The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) has been shown to be an endogenous counter-regulator of GC, and potentiates autoimmune-mediated neuroinflammation. In order to evaluate whether MIF levels are elevated in the cerebrospinal fluid (CSF) of MS patients (CSF-MIF), and whether they are higher still during a GC refractory relapse, we compared CSF-MIF concentrations of CIS/MS patients with acute optic neuritis as their first inflammatory episode (ON, n = 20), CIS/MS patients with a stable disease progression/without relapse (CIS/MS w/o, n = 18), and healthy controls (HC, n = 20) using ANOVA. Mean CSF-MIF concentrations in CIS/MS w/o patients were significantly higher than in ON patients and HCs, whereas ON patients and HCs did not differ. A subgroup analysis of the ON group revealed 10 patients to be responsive to GC-treatment (GC-ON) and 10 patients refractory under GC-treatment (rGC-ON). However, mean CSF-MIF concentrations did not differ between GC-ON and rGC-ON cases. We therefore conclude that MIF is not suitable for distinguishing GC responders from non-responders in a group of patients with acute optic neuritis, but it rather mirrors the ongoing inflammation in long-term MS disease progression. [ABSTRACT FROM AUTHOR]

The oral obligate anaerobe Porphyromonas gingivalis possesses a small conserved transcript PG_RS02100 of unknown function we previously identified using small RNA-seq analysis as expressed during logarithmic growth. In this study, we sought to determine if PG_RS02100 plays a role in P. gingivalis growth or stress response. We show that a PG_RS02100 deletion mutant’s (W83Δ514) ability to grow under anaerobic conditions was no different than wildtype (W83), but it was better able to survive hydrogen peroxide exposure when cultured under heme limiting growth conditions, and was more aerotolerant when plated on enriched whole blood agar and exposed to atmospheric oxygen. Together, these results indicate that PG_RS02100 plays a role in surviving oxidative stress in actively growing P. gingivalis and that P. gingivalis’ response to exogenous hydrogen peroxide stress is linked to heme availability. Relative qRT-PCR expression analysis of oxyR, trx-1, tpx, sodB, ahpC, dinF, cydB, and frd, in W83Δ514 and W83 in response to 1 h exogenous dioxygen or hydrogen peroxide exposure, when cultured with varying heme availability, support our phenotypic evidence that W83Δ514 has a more highly primed defense system against exogenous peroxide, dioxygen, and heme generated ROS. Interestingly, W83Δ514 turned black faster than W83 when cultured on whole blood agar, suggesting it was able to accumulate heme more rapidly. The mechanism of increased heme acquisition observed in W83Δ514 is not yet known. However, it is clear that PG_RS02100 is involved in modulating the P. gingivalis cell surface in a manner related to survival, particularly against oxidative stress. [ABSTRACT FROM AUTHOR]

GPIHBP1 is a protein localized at the endothelial cell surface that facilitates triglyceride (TG) lipolysis by binding lipoprotein lipase (LPL). Whether Glycosyl Phosphatidyl Inositol high density lipoprotein binding protein 1 (GPIHBP1) function is impaired and may underlie the hyperTG phenotype observed in type 2 diabetes is not yet established. To elucidate the mechanism underlying impaired TG homeostasis in insulin resistance state we studied the effect of insulin on GPIHBP1 protein expression in human microvascular endothelial cells (HMVEC) under flow conditions. Next, we assessed visceral adipose tissue GPIHBP1 protein expression in type 2 diabetes Lepr db/db mouse model as well as in subjects with ranging levels of insulin resistance. We report that insulin reduces the expression of GPIHBP1 protein in HMVECs. Furthermore, GPIHBP1 protein expression in visceral adipose tissue in Lepr db/db mice is significantly reduced as is the active monomeric form of GPIHBP1 as compared to Leprdb/m mice. A similar decrease in GPIHBP1 protein was observed in subjects with increased body weight. GPIHBP1 protein expression was negatively associated with insulin and HOMA-IR. In conclusion, our data suggest that decreased GPIHBP1 availability in insulin resistant state may hamper peripheral lipolysis capacity. [ABSTRACT FROM AUTHOR]

The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the peptides from background plasma contaminants and provided suitable response levels and linearity over an appropriate concentration range. Both compounds were found to be significantly degraded in rat plasma. Increasing degrees of both entrapment and stabilization were noted when dynantin was combined with the C14-alkyl-mannopyranoside in increasing peptide:glycoside ratios. The combination of MDP with the glycolipid also led to peptide entrapment, which greatly improved the plasma stability of the peptide. Overall, the results clearly indicate that the stability of peptide-based structures, which are subject to degradation in plasma, can be greatly improved via entrapment within C14-alkyl-mannopyranoside-bearing glycoliposomes. [ABSTRACT FROM AUTHOR]

Megakaryocytes (Mks) derive from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and develop into large, polyploid cells that eventually give rise to platelets. As Mks mature, they migrate from the bone marrow niche into the vasculature, where they are exposed to shear forces from blood flow, releasing Mk particles (platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs)) into circulation. We have previously shown that transcription factor p53 is important in Mk maturation, and that physiological levels of shear promote Mk particle generation and platelet biogenesis. Here we examine the role of p53 in the Mk shear-stress response. We show that p53 is acetylated in response to shear in both immature and mature Mks, and that decreased expression of deacetylase HDAC1, and increased expression of the acetyltransferases p300 and PCAF might be responsible for these changes. We also examined the hypothesis that p53 might be involved in the shear-induced Caspase 3 activation, phosphatidylserine (PS) externalization, and increased biogenesis of PLPs, PPTs, and MkMPs. We show that p53 is involved in all these shear-induced processes. We show that in response to shear, acetyl-p53 binds Bax, cytochrome c is released from mitochondria, and Caspase 9 is activated. We also show that shear-stimulated Caspase 9 activation and Mk particle biogenesis depend on transcription-independent p53-induced apoptosis (TIPA), but PS externalization is not. This is the first report to show that shear flow stimulates TIPA and that Caspase 9 activation and Mk-particle biogenesis are directly modulated by TIPA. [ABSTRACT FROM AUTHOR]

Computational models of normal liver function and xenobiotic induced liver damage are increasingly being used to interpret in vitro and in vivo data and as an approach to the de novo prediction of the liver’s response to xenobiotics. The microdosimetry (dose at the level of individual cells) of xenobiotics vary spatially within the liver because of both compound-independent and compound-dependent factors. In this paper, we build model liver lobules to investigate the interplay between vascular structure, blood flow and cellular transport that lead to regional variations in microdosimetry. We then compared simulation results obtained using this complex spatial model with a simpler linear pipe model of a sinusoid and a very simple single box model. We found that variations in diffusive transport, transporter-mediated transport and metabolism, coupled with complex liver sinusoid architecture and blood flow distribution, led to three essential patterns of xenobiotic exposure within the virtual liver lobule: (1) lobular-wise uniform, (2) radially varying and (3) both radially and azimuthally varying. We propose to use these essential patterns of exposure as a reference for selection of model representations when a computational study involves modeling detailed hepatic responses to xenobiotics. [ABSTRACT FROM AUTHOR]

The ability to restrict low molecular weight compounds to the gastrointestinal (GI) tract may enable an enhanced therapeutic index for molecular targets known to be associated with systemic toxicity. Using a triazolopyrazine CSF1R inhibitor scaffold, a broad range of prodrugs were synthesized and evaluated for enhanced delivery to the colon in mice. Subsequently, the preferred cyclodextrin prodrug moiety was appended to a number of CSF1R inhibitory active parent molecules, enabling GI-restricted delivery. Evaluation of a cyclodextrin prodrug in a dextran sodium sulfate (DSS)-induced mouse colitis model resulted in enhanced GI tissue levels of active parent. At a dose where no significant depletion of systemic monocytes were detected, the degree of pharmacodynamic effect–measured as reduction in macrophages in the colon–was inferior to that observed with a systemically available positive control. This suggests that a suitable therapeutic index cannot be achieved with CSF1R inhibition by using GI-restricted delivery in mice. However, these efforts provide a comprehensive frame-work in which to pursue additional gut-restricted delivery strategies for future GI targets. [ABSTRACT FROM AUTHOR]

Introduction: Ex vivo cell expansion under Good Manufacturing Practice (GMP) guidelines can be performed using medium additives containing human growth factors from platelets. These products can differently affect proliferation of adipose mesenchymal stromal stem cells (ASC). Qualification of medium additive performance is required for validation under GMP regulations: assessment of growth factor concentrations is not sufficient to predict the biological activity of the product batch. Proton nuclear magnetic resonance spectrometry (1H-NMR) and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) provide wide molecular characterization of samples. Aims: We aimed to assess if 1H-NMR and MALDI-TOF MS techniques can be used as quality control test potentially predicting the impact of a medium additive on cell proliferation. Methods: We tested the impact on ASC growth rate (cell proliferation assessment and cell morphology analysis) of four medium additives, obtained by different methods from human platelet apheresis product. In order to classify each medium additive, we evaluated growth factor concentrations and spectra obtained by 1H-NMR and by MALDI-TOF MS. Results: Medium additive obtained by CaCl2 activation of platelet rich products induced higher proliferation rate vs additive derived from platelet depleted ones. Additives obtained by freeze-and-thaw methods weakly induced ASC proliferation. As expected, principal component analysis of growth factor concentrations did not unravel specific biochemical features characterizing medium additives in relation with their biological activity. Otherwise, while 1H-NMR showed a partial resolution capacity, analysis of MALDI-TOF MS spectra allowed unambiguous distinction between the medium additives we used to differently stimulate cell growth in vitro. Discussion: MALDI-TOF and, despite limitations, 1H-NMR are promising cost effective and reliable quality controls to classify the potential of a medium additive to promote ASC growth. This can represent, after further investigations and appropriate validation, a significant advantage for GMP compliant manufacturing of advanced cell therapy products. [ABSTRACT FROM AUTHOR]

Background: Urine poses an attractive non-invasive means for obtaining liquid biopsies for oncological diagnostics. Especially molecular analysis on urinary DNA is a rapid growing field. However, optimal and practical storage conditions that result in preservation of urinary DNA, and in particular hypermethylated DNA (hmDNA), are yet to be determined. Aim: To determine the most optimal and practical conditions for urine storage that result in adequate preservation of DNA for hmDNA analysis. Methods: DNA yield for use in methylation analysis was determined by quantitative methylation specific PCR (qMSP) targeting the ACTB and RASSF1A genes on bisulfite modified DNA. First, DNA yield (ACTB qMSP) was determined in a pilot study on urine samples of healthy volunteers using two preservatives (Ethylenediaminetetraacetic acid (EDTA) and Urine Conditioning Buffer, Zymo Research) at four different temperatures (room temperature (RT), 4°C, -20°C, -80°C) for four time periods (1, 2, 7, 28 days). Next, hmDNA levels (RASSF1A qMSP) in stored urine samples of patients suffering from bladder cancer (n = 10) or non-small cell lung cancer (NSCLC; n = 10) were measured at day 0 and 7 upon storage with and without the addition of 40mM EDTA and/or 20 μl/ml Penicillin Streptomycin (PenStrep) at RT and 4°C. Results: In the pilot study, DNA for methylation analysis was only maintained at RT upon addition of preserving agents. In urine stored at 4°C for a period of 7 days or more, the addition of either preserving agent yielded a slightly better preservation of DNA. When urine was stored at -20 °C or -80 °C for up to 28 days, DNA was retained irrespective of the addition of preserving agents. In bladder cancer and NSCLC samples stored at RT loss of DNA was significantly less if EDTA was added compared to no preserving agents (p<0.001). Addition of PenStrep did not affect DNA preservation (p>0.99). Upon storage at 4°C, no difference in DNA preservation was found after the addition of preserving agents (p = 0.18). The preservation of methylated DNA (RASSF1A) was strongly correlated to that of unmethylated DNA (ACTB) in most cases, except when PCR values became inaccurate. Conclusions: Addition of EDTA offers an inexpensive preserving agent for urine storage at RT up to seven days allowing for reliable hmDNA analysis. To avoid bacterial overgrowth PenStrep can be added without negatively affecting DNA preservation. [ABSTRACT FROM AUTHOR]

Objectives: Malignancy is the strongest predictor for venous thromboembolism. Dual energy CT (DECT) can support assessment of pulmonary emboli (PE) by providing vessel reconstructions (DECT-VR) and highlighting thrombi. Purpose was to determine prevalence and risk factors of PE in staging examinations of stage IV-melanoma patients and to evaluate the potential of DECT-VR to improve PE diagnosis. Material and methods: This retrospective study was approved by IRB. Contrast-enhanced, conventional grey scale CT (cCT) and DECT-VR of 200 stage IV-melanoma patients were reviewed by three radiologists in consensus. Overall prevalence was determined. One-sided Wilcoxon-test was performed to compare the number of detected emboli between cCT and cCT with supplementary DECT-VR. Frequencies of risk factors were compared with χ2 test. Results: On cCT, 9 PE were detected (6 patients, correlating to 3% of the study population with 0.05 emboli per patient). With the supplementary DECT-VR, number of diagnosed emboli increased from 9 to 17 (p < 0.05) (in total 9 patients, correlating to 0.09 emboli per patient). Emboli on DECT-VR were mainly subsegmentally (7 of 8). There was no significant difference in the frequency of risk factors. Conclusions: The prevalence of pulmonary emboli in our cohort of 200 stage IV melanoma patients was 5%. DECT-VR improved significantly diagnosis of PE, especially when located subsegmentally. [ABSTRACT FROM AUTHOR]

At times when rhinoceros are fiercely poached, when some rhinoceros species are closer than ever to extinction, and when the scientific community is in debate over the use of advanced cell technologies as a remaining resort it is time to simplify and improve existing assisted reproduction techniques to enhance breeding and genetic diversity in the living populations under our care. Semen cryopreservation has been performed in all captive rhinoceros species with limited degree of success. Here we tested three freezing extenders, containing different cryoprotectants and various freezing rates for the cryopreservation of rhinoceros sperm from 14 bulls. In experiment I, semen from 9 bulls was used to determine the most suitable diluent, cryoprotectant and freezing rate for the successful cryopreservation of rhinoceros sperm. In experiment II, semen from 5 bulls was used to assess whether the removal of seminal plasma could further improve post thaw sperm quality following cryopreservation with conditions identified in Experiment I. Semen was diluted with Berliner Cryomedia, ButoCrio® or INRA Freeze®, packaged in 0.5 mL straws and frozen 3, 4, and 5 cm over liquid nitrogen (LN) vapour or directly in a dryshipper. It was found that semen extended with ButoCrio® (containing glycerol and methylformamide) and frozen 3cm over LN vapour provided the best protection to rhinoceros spermatozoa during cryopreservation. When pooled over treatments, total and progressive post thaw motility was 75.3 ± 4.2% and 68.5 ± 5.7%, respectively marking a new benchmark for the cryopreservation of rhinoceros sperm. Post thaw total and progressive motility, viability and acrosome integrity of semen diluted in ButoCrio® was significantly higher than semen extended in Berliner Cryomedia or INRA Freeze®. The removal of seminal plasma did not improve post thaw sperm survival (p > 0.05). In conclusion, the cryosurvival of rhinoceros spermatozoa was significantly improved when using a mixture of glycerol and methylformamide in combination with a fast freezing rate at 3 cm. These results describe a new protocol for the improved cryosurvival of rhinoceros spermatozoa and will enable a more successful preservation of genetic diversity between males, especially in donors whose spermatozoa may already be compromised prior to or during collection. The successful reduction of glycerol concentration in favour of methylformamide as a cryoprotectant could be a novel suggestion for the improvement of cryopreservation techniques in other wildlife species. [ABSTRACT FROM AUTHOR]

Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood ex vivo. However, the sources of the nitrosylating species in vivo and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO2; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors ex vivo, while it was enhanced upon caveolin-1 deletion in vivo, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function. [ABSTRACT FROM AUTHOR]

This study aimed to identify clinical or microbiological factors affecting the clinical relevance of Corynebacterium striatum isolated from blood cultures. A total of 64 isolates from 51 patients identified as C. striatum by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing were assessed. More than two blood cultures were positive in 25 (48.1%) patients. Diabetes, solid tumor, and a history of previous exposure to antibiotics were more common in patients with multiple positive blood cultures. Charlson comorbidity scores were also higher, and more isolates were recovered after 48 hours of hospital stay in patients with multiple positive blood cultures. Strains recovered from patients with multiple positive blood cultures produced significantly more biofilm. Based on multilocus sequence typing (MLST), sequence type (ST) 20 (31.3%) was the most dominant, followed by ST2 (20.3%) and ST23 (10.9%). There was no relationship between the number of positive blood culture sets and sequence typing. In multivariate analyses, Carlson comorbidity score (odds ratio [OR], 1.91; 95% confidence interval [CI], 1.09–3.36; P = 0.03) and biofilm formation were associated with multiple positive blood cultures (OR, 17.43; 95% CI, 3.71–81.91; P = 0.03). This study provides evidence that the biofilm phenotype could contribute to determining the clinical significance of C. striatum in patients with severe underlying conditions. The predominance of certain STs suggests the relatedness of C. striatum infection and the nosocomial environment. [ABSTRACT FROM AUTHOR]

Arctigenin is evaluated for antitumor efficacy in patients with pancreatic cancer. It has an inhibitory activity on mitochondrial complex I.Therefore, plasma lactate level of patients after arctigenin administration was evaluated for biomarker of clinical response and/or adverse effect. Plasma lactate level in 15 patients enrolled in a Phase I clinical trial of GBS-01 rich in arctigenin was analyzed by colorimetric assay. Statistical analyses for association of plasma lactate and clinical responses, pharmacokinetics of arctigenin, and background factors of each patient by multivariate and univariate analyses.In about half of the patients, transient increase of lactate was observed. Correlation between plasma lactate level and pharmacokinetic parameters of arctigenin and its glucuronide conjugate, and clinical outcome was not detected. Regarding to the determinant of lactate level, only slight association with liver function test was detected. Plasma lactate level is primary determined by reutilization rather than production for antitumor effect and dose not serve as a biomarker. Arctigenin, inhibition of mitochondrial complex I, plasma lactate concentration, phase I clinical trial of GBS-01, Cori cycle. [ABSTRACT FROM AUTHOR]

While the cerebral autoregulation sufficiently protects subcortical brain regions during hypoxia or asphyxia, the cerebral cortex is not as adequately protected, which suggests that regulation of the cerebral blood flow (CBF) is area-specific. Hypoxia was induced by inhalation of 5% oxygen, for reoxygenation 100% oxygen was used. Cortical and subcortical CBF (by laser Doppler flowmetry), blood gases, mean arterial blood pressure (MABP), heart rate and renal blood flow were constantly monitored. Low dosed urapidil was used for α1A-adrenergic receptor blockade. Western blotting was used to determine adrenergic receptor signalling mediators in brain arterioles. During hypoxia cortical CBF decreased to 72 ± 11% (mean reduction 11 ± 3%, p < 0.001) of baseline, whereas subcortical CBF increased to 168±18% (mean increase 43 ± 5%, p < 0.001). Reoxygenation led to peak CBF of 194 ± 27% in the subcortex, and restored cortical CBF. α1A-Adrenergic blockade led to minor changes in cortical CBF, but massively reduced subcortical CBF during hypoxia and reoxygenation–almost aligning CBF in both brain regions. Correlation analyses revealed that α1A-adrenergic blockade renders all CBF-responses pressure-passive during hypoxia and reoxygenation, and confirmed the necessity of α1A-adrenergic signalling for coupling of CBF-responses to oxygen saturation. Expression levels and activation state of key signalling-mediators of α1-receptors (NOSs, CREB, ERK1/2) did not differ between cortex and subcortex. The dichotomy between subcortical and cortical CBF during hypoxia and reoxygenation critically depends on α1A-adrenergic receptors, but not on differential expression of signalling-mediators: signalling through the α1A-subtype is a prerequisite for cortical/subcortical redistribution of CBF. [ABSTRACT FROM AUTHOR]

Recent studies suggest that aberrant DNA methylation might occur early and commonly in colorectal tumorigenesis. In 111 normal subjects, the mean LINE-1 methylation level of peripheral blood was 81.0 ± 5.7%. Of 143 colorectal cancer (CRC) patients, the mean level of LINE-1 methylation was 60.5 ± 12.5%. We defined below 60% as cut-off value of LINE-1 hypomethylation, and 93 cases (65.0%) had LINE-1 hypomethylation in the tumor tissue. LINE-1 hypomethylation was not associated with any other clinical features. There was a trend that LINE-1 hypomethylation tumors were associated with advanced disease, but it did not reach statistical significance. There was no significant association between mutations of 12 genes, MSI-high, EMAST, and LINE-1 hypomethylation level. The median follow-up was 61.2 months. Five-year disease-free survival (DFS) and overall survival curves of patients with LINE-1 hypomethylation tumors were significantly lower than those of patients with normal LINE-1 methylation tumors (p = 0.032 and 0.001, respectively). Multivariate analysis showed that only TNM staging was an independent prognostic factor for CRC patients including DFS and overall survival (OS). LINE-1 did not impact patients’ outcomes in multivariate analysis including DFS and OS. In conclusion, LINE-1 hypomethylation is marginally related to advanced stage CRC and impacts patients’ outcomes in univariate analysis. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study is to identify potential CpG site(s) or DNA methylation pattern(s) in the pseudo α-globin 1 gene (HBAP1 gene), the gene which locates in α-thalassemia-1 deletion mutation, to differentiate plasma DNA between pregnant and non-pregnant women. Method: DNA methylation profiles of placenta and peripheral blood from the MethBase database were compared to screen differentially methylated regions. This region was confirmed the differential by methylation-sensitive high resolution melt (MS-HRM) analysis. The differential region was used to compare DNA methylation profile of plasma DNA between pregnant and non-pregnant women by bisulfite amplicon sequencing in three levels: overall, individual CpG sites and individual molecules (DNA methylation patterns). Result: Using MethBase data, four consecutive CpG sites in the HBAP1 gene were identified as regions of differential DNA methylation between placenta and peripheral blood. The confirmation by MS-HRM showed the differential DNA methylation profile between the placenta and plasma from non-pregnant women. The comparison of DNA methylation profiles between the plasma of pregnant and non-pregnant women showed that, in the overall levels of the four CpG sites, DNA methylation of pregnant women was detected at lower levels than non-pregnant women. In the individual CpG site level, only the second CpG site showed differential DNA methylation between the groups. In the DNA methylation pattern level, there was no strongly significant differences in DNA methylation patterns between the pregnant and non-pregnant groups. Conclusion: Our result demonstrated that, in the plasma from pregnant women, only one of the four CpG sites displays a decrease in DNA methylation compared with non-pregnant women. It indicates that this CpG site might be useful for determining the presence or absence of fetal wild-type α-globin gene cluster allele in maternal plasma. [ABSTRACT FROM AUTHOR]

Ossification of the posterior longitudinal ligament (OPLL), a spinal ligament, reduces the range of motion in limbs. No treatment is currently available for OPLL, which is why therapies are urgently needed. OPLL occurs in obesity, is more common in men, and has an onset after 40 years of age. The mechanisms underlying OPLL remain unclear. In this study, we performed a serum proteomic analysis in both OPLL patients and healthy subjects to identify factors potentially involved in the development of OPLL, and found reduced levels of a protein that might underlie the pathology of OPLL. We isolated the protein, determined its amino acid sequence, and identified it as chemokine (C-X-C motif) ligand 7 (CXCL7). Based on these proteomics findings, we generated a CXCL7 knockout mouse model to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossification in the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7 deficiency in OPLL, we searched for single nucleotide polymorphisms and altered DNA exons, but no abnormalities were found. Although miR-340 levels were found to be high in an miRNA array, they were insufficient to reduce CXCL7 levels. Ubiquitin C-terminal hydrolase1 (UCHL1) was found to be overexpressed in CXCL7-null mice and in the sera of patients with OPLL, and was correlated with OPLL severity. Post-translational modifications of proteins with ubiquitin and ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) enzymes, are thought to control a wide range of cellular processes, and alterations in the ubiquitin–proteasome system have been associated with several degenerative disorders. In addition, the OPLL tissue of CXCL7-null mouse and its primary cells expressed the antibody to ubiquitin (linkage-specific K48). Our data clearly show decreased CXCL7 levels in patients with OPLL, and that OPLL developed in mice lacking CXCL7. Tumor necrosis factor receptor-associated factor (TRAF)6 expression was decreased in CXCL7-null mouse primary cells. Furthermore, K48 polyubiquitination was found in posterior longitudinal ligament ossified tissue and primary cells from CXCL7-null mice. We performed a phosphoproteomics analysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase (ME3K)15, ubiquitin protein ligase E3C (UBE3C) and protein kinase C (PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7 deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitination in the onset of OPLL. In conclusion, CXCL7 levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7 levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL. [ABSTRACT FROM AUTHOR]

Background: In fish with external fertilization, two main start-up mechanisms of the path that blocks or activates the spermatozoan motility apparatus are known. The main factor managing the path is osmolality or potassium ion. In burbot from the European and North American population, contradictory findings regarding the factors influencing the onset of spermatozoa motility were reported. The objective of the current study was to determine the effect of potassium and osmolality on the spermatozoa activation of European burbot, Lota lota (Actinopterygii, Gadiformes, Lotidae). Moreover, the influence of pH, as well as sodium ion concentrations on spermatozoa motility was investigated. Seven parameters characterising motility were traced by means of computer-assisted sperm analysis (CASA). Principal findings: The spermatozoa of European burbot are K+ ion-sensitive. A 6-mM KCl solution significantly decreased motility, and above 12-mM (50 mOsm kg-1) totally ceased spermatozoa movement. Sucrose and Na+ solutions inhibited spermatozoa movement only at concentrations > 450–480 mOsm kg-1. Greater differences in the percentage of motile sperm between individuals were noted in solutions containing high concentrations of chemicals triggering sperm motility. The optimum osmolality for spermatozoa motility is in the range of 100–200 mOsm kg-1. The burbot spermatozoa were motile over a wide range of pH values with the best activation at pH 9. Conclusion: It was demonstrated that the spermatozoa of European burbot are inhibited by K+ ions similarly as in North American burbot. Other electrolyte and non-electrolyte solutions inhibit spermatozoa movement only if their osmolality is greater than that of the physiological osmolality of seminal plasma. The data provided on basic knowledge of burbot spermatozoa allow to ensure appropriate conditions during artificial reproduction and scientific research. [ABSTRACT FROM AUTHOR]

One of the most ambitious endeavors in the field of diabetes technology is non-invasive glucose sensing. In the past decades, a number of different technologies have been assessed, but none of these have found its entry into general clinical use. We report on the development of a table-top confocal Raman spectrometer that was used in the home of patients with diabetes and operated for extended periods of time unsupervised and without recalibration. The system is based on measurement of glucose levels at a ‘critical depth’ in the skin, specifically in the interstitial fluid located below the stratum corneum but above the underlying adipose tissue layer. The region chosen for routine glucose measurements was the base of the thumb (the thenar). In a small clinical study, 35 patients with diabetes analyzed their interstitial fluid glucose for a period of 60 days using the new critical-depth Raman (CD-Raman) method and levels were correlated to reference capillary blood glucose values using a standard finger-stick and test strip product. The calibration of the CD-Raman system was stable for > 10 days. Measurement performance for glucose levels present at, or below, a depth of ~250μm below the skin surface was comparable to that reported for currently available invasive continuous glucose monitors. In summary, using the CD-Raman technology we have demonstrated the first successful use of a non-invasive glucose monitor in the home. [ABSTRACT FROM AUTHOR]

Background: Mercury (Hg) is a well-recognized environmental pollutant known by its toxicity of development and neurotoxicity, which results in adverse health outcomes. However, the mechanisms underlying the teratogenic effects of Hg are not well understood. Imprinting genes are emerging regulators for fetal development subjecting to environmental pollutants impacts. In this study, we examined the association between preconceptional Hg exposure and the alteration of DNA methylation of imprinting genes H19, Meg3, and Peg3 in human sperm DNA. Methods: A total of 616 men, aged from 22 to 59, were recruited from Reproductive Medicine Clinic of Maternal and Child Care Service Center and the Urologic Surgery Clinic of Shanxi Academy of Medical Sciences during April 2015 and March 2016. Demographic information was collected through questionnaires. Urine was collected and urinary Hg concentrations were measured using a fully-automatic double-channel hydride generation atomic fluorescence spectrometer. Methylation of imprinting genes H19, Meg3 and Peg3 of sperm DNA from 242 participants were examined by bisulfite pyrosequencing. Spearman’s rank and multivariate regression analysis were used for correlation analysis between sperm DNA methylation status of imprinting genes and urinary Hg levels. Results: The median concentration of Hg for 616 participants was 9.14μg/l (IQR: 5.56–12.52 μg/l; ranging 0.16–71.35μg/l). A total of 42.7% of the participants are beyond normal level for non-occupational exposure according to the criterion of Hg poisoning (≥10 μg/L). Spearman’s rank analysis indicated a negative correlation between urinary Hg concentrations and average DNA methylation levels of imprinted genes H19 (rs = −0.346, p <0.05), but there was no such a correlation for Peg3 and Meg3. Further, we analyzed the correlation between methylation level at individual CpG site of H19 and urinary Hg level. The results showed a negative correlation between urinary Hg concentrations and three out of seven CpG sites on H19 DMR, namely CpG2 (rs = −0.137, p <0.05), CpG4 (rs = −0.380, p <0.05) and CpG6 (rs = −0.228, p <0.05). After adjusting age, smoking, drinking, intake of aquatic products and education by multivariate regression analysis, the results have confirmed the correlation as mentioned above. Conclusions: Mercury non-occupational environmental exposure in reproductive-aged men was associated with altered DNA methylation outcomes at imprinting gene H19 in sperm, implicating the susceptibility of the developing sperm for environmental insults. [ABSTRACT FROM AUTHOR]

Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood. Antibody variants with optimal pH-dependent binding to C5 exhibited little to no TMDD in mice in the presence of human C5. To further enhance the efficiency of FcRn-mediated recycling of the antibody, two additional substitutions were introduced to increase affinity for human FcRn. These substitutions yielded an additional doubling of the t½ of surrogate anti-mouse C5 antibodies with reduced TMDD in transgenic mice expressing the human FcRn. In conclusion, ALXN1210 is a promising new therapeutic candidate currently in clinical development for treatment of patients with PNH and atypical hemolytic uremic syndrome. [ABSTRACT FROM AUTHOR]

Rationale: Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. Objective: To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. Methods and results: The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. Conclusion: NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism. [ABSTRACT FROM AUTHOR]

Aim: To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. Background: DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. Methods: QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Results: Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. Conclusion: High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease. [ABSTRACT FROM AUTHOR]

Epigenetics is believed to provide great chances for a better understanding of the development and treatment of many diseases where the analysis of genomic DNA has so far failed to provide conclusive answers. Methylcytosine is a frequently used quantitative marker of epigenetic studies. Since immediate analysis of sampled material is in most cases not possible, storage time and conditions are critical aspects regarding the quality of genomic DNA and reliability of analysis. Blood is frequently used for such analyses. We, therefore, collected blood samples of ten volunteers and stored them under various conditions for ten months: -70°C, -20°C, 2–8°C and room temperature. An additional aliquot was frozen at -70°C and thawed once a week at room temperature. We then compared the DNA extraction yields and methylation status in relation to storage time and conditions. We found significantly lower DNA extraction yields (up to -97.45%; p ≤ 0.001) as well as significantly higher methylation levels after ten months of storage (up to +42.0%; p ≤ 0.001). These results suggest that storage time has an important influence on DNA analyses of blood samples for all storage conditions. This might be due to differences in stability of methylated and non-methylated DNA. Our study indicates that storage conditions and time may be a critical factor for epigenetic methylation studies and require rigorous validation. For reliable analyses we, therefore, recommend to perform epigenetic analysis directly after sample collection. [ABSTRACT FROM AUTHOR]