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Background: Dietary management is highly important for the maintenance of renal function in patients with chronic kidney disease (CKD). Cerebral oxygen saturation (rSO2) was reportedly associated with the estimated glomerular filtration rate (eGFR) and cognitive function. However, data concerning the association between cerebral rSO2 and dietary intake of CKD patients is limited. Methods: This was a single-center observational study. We recruited 67 CKD patients not undergoing dialysis. Cerebral rSO2 was monitored using the INVOS 5100c oxygen saturation monitor. Energy intake was evaluated by dietitians based on 3-day meal records. Daily protein and salt intakes were calculated from 24-h urine collection. Results: Multivariable regression analysis showed that cerebral rSO2 was independently associated with energy intake (standardized coefficient: 0.370) and serum albumin concentration (standardized coefficient: 0.236) in Model 1 using parameters with p < 0.10 in simple linear regression analysis (body mass index, Hb level, serum albumin concentration, salt and energy intake) and confounding factors (eGFR, serum sodium concentration, protein intake), and the energy/salt index (standardized coefficient: 0.343) and Hb level (standardized coefficient: 0.284) in Model 2 using energy/protein index as indicated by energy intake/protein intake and energy/salt index by energy intake/salt intake in place of salt, protein and energy intake. Conclusions: Cerebral rSO2 is affected by energy intake, energy/salt index, serum albumin concentration and Hb level. Sufficient energy intake and adequate salt restriction is important to prevent deterioration of cerebral oxygenation, which might contribute to the maintenance of cognitive function in addition to the prevention of renal dysfunction in CKD patients. [ABSTRACT FROM AUTHOR]

Recent experimental work has revealed unusual features of the effect of certain drugs on cardiac inwardly rectifying potassium currents, including the constitutively active and acetylcholine-induced components of acetylcholine-sensitive current (IKAch). These unusual features have included alternating susceptibility of the current components to activation and inhibition induced by ethanol or nicotine applied at various concentrations, and significant correlation between the drug effect and the current magnitude measured under drug-free conditions. To explain these complex drug effects, we have developed a new type of quantitative model to offer a possible interpretation of the effect of ethanol and nicotine on the IKAch channels. The model is based on a description of IKAch as a sum of particular currents related to the populations of channels formed by identical assemblies of different α-subunits. Assuming two different channel populations in agreement with the two reported functional IKAch-channels (GIRK1/4 and GIRK4), the model was able to simulate all the above-mentioned characteristic features of drug-channel interactions and also the dispersion of the current measured in different cells. The formulation of our model equations allows the model to be incorporated easily into the existing integrative models of electrical activity of cardiac cells involving quantitative description of IKAch. We suppose that the model could also help make sense of certain observations related to the channels that do not show inward rectification. This new ionic channel model, based on a concept we call population type, may allow for the interpretation of complex interactions of drugs with ionic channels of various types, which cannot be done using the ionic channel models available so far. [ABSTRACT FROM AUTHOR]

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]

The developmental speed of new antimicrobials does not meet the emergence of multidrug-resistant bacteria sufficiently. A potential shortcut is assessing the antimicrobial activity of already approved drugs. Intrudingly, the antibacterial action of glatiramer acetate (GA) has recently been discovered. GA is a well-known and safe immunomodulatory drug particular effective against Gram-negative bacteria, which disrupts biological membranes by resembling the activity of antimicrobial peptides. Thus, GA can potentially be included in treatment strategies used to combat infections caused by multidrug-resistant Gram-negatives. One potential application is chronic respiratory infections caused by Pseudomonas aeruginosa, however the safety of GA inhalation has never been assessed. Here, the safety of inhaling nebulized GA is evaluated in a preclinical pig model. The potential side effects, i.e., bronchoconstriction, respiratory tract symptoms and systemic- and local inflammation were assessed by ventilator monitoring, clinical observation, biochemistry, flowcytometry, and histopathology. No signs of bronchoconstriction assessed by increased airway peak pressure, Ppeak, or decreased oxygen pressure were observed. Also, there were no signs of local inflammation in the final histopathology examination of the pulmonary tissue. As we did not observe any potential pulmonary side effects of inhaled GA, our preliminary results suggest that GA inhalation is safe and potentially can be a part of the treatment strategy targeting chronic lung infections caused by multidrug-resistant Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated. [ABSTRACT FROM AUTHOR]

Two novel and related pathogenic variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene were structurally and functionally characterized. These alterations have not been previously described in literature. Two patients with diagnosis of Cystic Fibrosis (CF) based on the presence of one mutated allele, p.Phe508del, pathological sweat test and clinical symptoms were studied. To complete the genotypes of both patients, an extensive genetic and functional analysis of the CFTR gene was performed. Extensive genetic characterization confirmed the presence of p.Phe508del pathogenic variant and revealed, in both patients, the presence of an insertion of part of intron 10 in intron 9 of the CFTR gene, within the (TG)m repeat, with a variable poly-T stretch. The molecular lesions resulted to be very similar in both patients, with only a difference in the number of T in the poly-T stretch. The functional characterization at RNA level revealed a complete anomalous splicing, without exon 10, from the allele with the insertion of both patients. Consequently, the alleles with the insertions are expected not to contribute to the formation of a functional CFTR protein. Molecular and functional features of these alterations are compatible with the definition of novel CF-causing variants of the CFTR gene. This also allowed the completion of the genetic characterization of both patients. [ABSTRACT FROM AUTHOR]

Background: This study investigated the incidence and predictors of retreatment after discontinuation of either entecavir (ETV) or tenofovir disoproxil fumarate (TDF) treatment in Taiwan. Methods: A total of 535 non-cirrhotic chronic hepatitis B (CHB) patients undergoing either ETV (n = 358) or TDF (n = 177) treatment were enrolled. Patients were followed for at least 12 months after stopping ETV or TDF treatment. Most patients (86.3%) fulfilled the retreatment criteria of Taiwan's National Health Plan. Results: The 5-year cumulative rates of clinical relapse and retreatment were 52.1% and 47%, respectively, in 160 hepatitis B e antigen (HBeAg)-positive patients, and were 62% and 54.8%, respectively, in 375 HBeAg-negative patients. The median duration from the end of treatment until clinical relapse and retreatment was 40 and 57 weeks, respectively, for all patients. Multivariate Cox regression analysis revealed that discontinuing TDF treatment, old age, male gender, and higher baseline HBsAg levels were independent factors of retreatment in HBeAg-positive patients; old age, HBV genotype B, and higher baseline and end-of-treatment HBsAg levels were independent factors in HBeAg-negative patients. A total of 18.8% of retreated patients satisfied the retreatment criteria of hepatic decompensation according to Taiwan's National Health Plan. Of the 64 patients who had clinical relapse without retreatment, 17 achieved sustained virological remission and 26 did not experience clinical relapse until their last visit after clinical relapse. Four patients developed HBsAg loss. Conclusions: The 5-year retreatment rate was about 50% in HBeAg-positive and HBeAg-negative patients. Discontinuing TDF treatment was an independent factor of retreatment in HBeAg-positive patients. [ABSTRACT FROM AUTHOR]

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3’,5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS. [ABSTRACT FROM AUTHOR]

Concentrations of free amino acids and [K+] in human sweat can be many times higher than in plasma. Conversely, [Na+] and [Cl-] in sweat are hypotonic to plasma. It was hypothesised that the amino acids and K+ were directly or indirectly associated with the resorption of Na+ and Cl- in the sweat duct. The implication would be that, as resources of these components became limiting during prolonged exercise then the capacity to resorb [Na+] and [Cl-] would diminish, resulting in progressively higher levels in sweat. If this were the case, then [Na+] and [Cl-] in sweat would have inverse relationships with [K+] and the amino acids during exercise. Forearm sweat was collected from 11 recreational athletes at regular intervals during a prolonged period of cycling exercise after 15, 25, 35, 45, 55 and 65 minutes. The subjects also provided passive sweat samples via 15 minutes of thermal stimulation. The sweat samples were analysed for concentrations of amino acids, Na+, Cl-, K+, Mg2+ and Ca2+. The exercise sweat had a total amino acid concentration of 6.4 ± 1.2mM after 15 minutes which was lower than the passive sweat concentration at 11.6 ± 0.8mM (p<0.05) and showed an altered array of electrolytes, indicating that exercise stimulated a change in sweat composition. During the exercise period, [Na+] in sweat increased from 23.3 ± 3.0mM to 34.6 ± 2.4mM (p<0.01) over 65 minutes whilst the total concentrations of amino acids in sweat decreased from 6.4 ± 1.2mM to 3.6 ± 0.5mM. [Na+] showed significant negative correlations with the concentrations of total amino acids (r = -0.97, p<0.05), K+ (r = -0.93, p<0.05) and Ca2+ (r = -0.83, p<0.05) in sweat. The results supported the hypothesis that amino acids and K+, as well as Ca2+, were associated with resorption of Na+ and Cl-. [ABSTRACT FROM AUTHOR]

Raman chemometric urinalysis (Rametrix™) was used to analyze 235 urine specimens from healthy individuals. The purpose of this study was to establish the “range of normal” for Raman spectra of urine specimens from healthy individuals. Ultimately, spectra falling outside of this range will be correlated with kidney and urinary tract disease. Rametrix™ analysis includes direct comparisons of Raman spectra but also principal component analysis (PCA), discriminant analysis of principal components (DAPC) models, multivariate statistics, and it is available through GitHub as the Rametrix™ LITE Toolbox for MATLAB®. Results showed consistently overlapping Raman spectra of urine specimens with significantly larger variances in Raman shifts, found by PCA, corresponding to urea, creatinine, and glucose concentrations. A 2-way ANOVA test found that age of the urine specimen donor was statistically significant (p < 0.001) and donor sex (female or male identification) was less so (p = 0.0526). With DAPC models and blind leave-one-out build/test routines using the Rametrix™ PRO Toolbox (also available through GitHub), an accuracy of 71% (sensitivity = 72%; specificity = 70%) was obtained when predicting whether a urine specimen from a healthy unknown individual was from a female or male donor. Finally, from female and male donors (n = 4) who contributed first morning void urine specimens each day for 30 days, the co-occurrence of menstruation was found statistically insignificant to Rametrix™ results (p = 0.695). In addition, Rametrix™ PRO was able to link urine specimens with the individual donor with an average of 78% accuracy. Taken together, this study established the range of Raman spectra that could be expected when obtaining urine specimens from healthy individuals and analyzed by Rametrix™ and provides the methodology for linking results with donor characteristics. [ABSTRACT FROM AUTHOR]

We identified a mouse strain, HLB444, carrying an N-ethyl-N-nitrosourea (ENU)-induced mutation in a highly conserved C2H2 zinc-finger DNA binding motif of the transcriptional regulator KLF15 that exhibits resistance to diet-induced obesity. Characterization of the HLB444 mutant model on high-fat and chow diets revealed a number of phenotypic differences compared to wild-type controls. When fed a high fat diet, HLB444 had lower body fat, resistance to hepatosteatosis, lower circulating glucose and improved insulin sensitivity compared to C57BL/6J controls. Gut microbial profiles in HLB444 generated from 16S rRNA sequencing of fecal samples differed from controls under both chow and high fat diets. HLB444 shares similar phenotypic traits with engineered full- and adipose-specific Klf15 knockout strains; however, some phenotypic differences between this mutant and the other models suggest that the Klf15 mutation in HLB444 is a hypomorphic variant. The HLB444 model will inform further annotation of transcriptional functions of KLF15, especially with respect to the role of the first zinc-finger domain. [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]

We previously reported that allogeneic, intraperitoneally administered “Neo-Islets,” composed of cultured pancreatic islet cells co-aggregated with high numbers of immunoprotective and cytoprotective Adipose-derived Stem Cells, reestablished, through omental engraftment, redifferentiation and splenic and omental up-regulation of regulatory T-cells, normoglycemia in autoimmune Type-1 Diabetic Non-Obese Diabetic (NOD) mice without the use of immunosuppressive agents or encapsulation devices. Based on these observations, we are currently testing this Neo-Islet technology in an FDA guided pilot study (INAD 012–776) in insulin-dependent, spontaneously diabetic pet dogs by ultrasound-guided, intraperitoneal administration of 2x10e5 Neo-Islets/kilogram body weight to metabolically controlled (blood glucose, triglycerides, thyroid and adrenal functions) and sedated animals. We report here interim observations on the first 4 canine Neo-Islet-treated, insulin-dependent pet dogs that are now in the early to intermediate-term follow-up phase of the planned 3 year study (> 6 months post treatment). Current results from this translational study indicate that in dogs, Neo-Islets appear to engraft, redifferentiate and physiologically produce insulin, and are rejected by neither auto- nor allo-immune responses, as evidenced by (a) an absent IgG response to the allogeneic cells contained in the administered Neo-Islets, and (b) progressively improved glycemic control that achieves up to a 50% reduction in daily insulin needs paralleled by a statistically significant decrease in serum glucose concentrations. This is accomplished without the use of anti-rejection drugs or encapsulation devices. No adverse or serious adverse events related to the Neo-Islet administration have been observed to date. We conclude that this minimally invasive therapy has significant translational relevance to veterinary and clinical Type 1 diabetes mellitus by achieving complete and at this point partial glycemic control in two species, i.e., diabetic mice and dogs, respectively. [ABSTRACT FROM AUTHOR]

In the course of the Maillard reaction, which occurs during heating of food but also under physiological condition, a broad spectrum of reaction products is formed. Among them, the advanced glycation endproducts (AGEs) Nε-carboxymethyllysine (CML), pyrraline (Pyr), methylglyoxal-derived hydroimidazolone 1 (MG-H1) and Nε-carboxyethyllysine (CEL) are the quantitatively dominating compounds during later reaction stages. Those dietary glycation compounds are under discussion as to be associated with chronic inflammation and the pathophysiological consequences of diseases such as diabetes. In the present study, the concentration of individual glycation compounds in saliva was monitored for the first time and related to their dietary uptake. Fasting saliva of 33 metabolically healthy subjects was analyzed with HPLC-MS/MS. The observed levels of individual glycation compounds ranged from 0.5 to 55.2 ng/ml and differed both intra- and interindividually. Patterns did not correlate with subject-related features such as vegetarianism or sports activities, indicating that dietary intake may play an important role. Therefore, six volunteers were asked to eat a raw food diet free of glycation compounds for two days. Within two days, salivary Pyr was lowered from median 1.7 ng/ml to a minimum level below the limit of detection, and MG-H1 decreased from 3.6 to 1.7 ng/ml in in a time-dependent manner after two days. Salivary CML and CEL concentrations were not affected. Therefore, measuring Pyr and MG-H1 in saliva is a suitable diagnostic tool to monitor the dietary intake and metabolic transit of glycation compounds present in heated foods. [ABSTRACT FROM AUTHOR]

Feed accounts for the greatest proportion of egg production costs and there is substantial variation in feed to egg conversion ratio (FCR) efficiency between individual hens. Despite this understanding, there is a paucity of information regarding layer hen feeding behaviour, diet selection and its impact on feed efficiency. It was hypothesised that variation in feed to egg conversion efficiency between hens may be influenced by feeding behaviour. For this experiment, two 35-bird groups of ISA Brown layers were selected from 450 individually caged hens at 25–30 weeks of age for either low FCR < 1.8 ± 0.02 (high feed efficiency (HFE) or high FCR > 2.1 ± 0.02 (low feed efficiency (LFE)). For each of these 70 hens, intake of an ad-libitum mash diet at 2-minute time intervals, 24 h a day, for 7 days was determined alongside behavioural assessment and estimation of the selection of components of the mash. The group selected for HFE had a lower feed intake, similar egg mass and associated lower FCR when compared with the LFE group. Whilst feed intake patterns were similar between HFE and LFE hens, there was a distinct intake pattern for all layer hens with intake rate increasing from 0300 to 1700 h with a sharp decline to 2200 h. High feed efficiency hens selected a diet with 25% more ash and 4% less gross energy than LFE hens. The LFE hens also spent more time eating with more walking events, but less time spent resting, drinking, preening and cage pecking events as compared with HFE hens. In summary, there was no contrasting diurnal pattern of feed consumption behaviour between the groups ranked on feed efficiency, however high feed efficiency hens consumed less feed and selected a diet with greater ash content and lower gross energy as compared with LFE hens. Our work is now focused on individual hen diet selection from mash diets with an aim of formulating precision, targeted diets for greater feed efficiency. [ABSTRACT FROM AUTHOR]

Background: As a recognised effective and economical agent for dental caries prevention, fluoride has been used in many different fluoridation schemes implemented across the world. Considering the narrow ‘dose-gap’ between the benefit of caries reduction and the risk of dental fluorosis, it is recommended that fluoride intake is monitored by measuring urinary fluoride excretion. The aim of this scoping review is to map the current literature/evidence on fluoride intake and excretion studies in relation to the study population, settings, type of study design, methodology, and analytical approach. Methods: Embase/Ovid, MEDLINE/Ovid, CINAHL/EBSCO, Scopus/Elsevier were searched for relevant articles until April 2018. Studies were included if they reported intake and excretion of fluoride in healthy humans of all age groups. Findings were explored using a narrative synthesis to summarise studies characteristics and outcome measures. Results: Removal of duplicates from the originally 2295 identified records yielded 1093 studies of which 206 articles were included. Only 21.6% of the studies were conducted in children (<8-year-olds). Most studies (38.8%) used drinking water concentration as a proxy for fluoride intake, whereas only 11.7% measured fluoride intake from all sources. Of the 72 studies that measured dietary fluoride intake, only 10 reported the validity of the employed dietary assessment method. Only 14 studies validated the urine sample collection methods. No information on the validity of the employed analytical method was reported by the majority (64.6%) of studies. Only a small proportion (8.7%) of the included studies investigated the association between fluoride intake and excretion. Conclusion: The findings reveal much variability in terms of conducting the studies and reporting the findings, illustrating a high heterogeneity in data collection across settings and populations. Future studies should provide more detail on sampling technique, measurement protocols (including validation), and on clearly defining the relationship between intake and urinary excretion of fluoride. [ABSTRACT FROM AUTHOR]

Comparing the efficacy of inhaled antibiotics can be difficult in small groups of patients with cystic fibrosis and mild lung disease. In a feasibility study we compared Aztreonam lysine for inhalation solution (AZLI; Cayston®) to standard inhaled antibiotic therapy in patients with cystic fibrosis and near normal spirometry. To detect treatment responses we used both lung clearance index (LCI) and forced expiratory volume in one second (FEV1). At baseline, median FEV1 was 87% pred. and median LCI was 8.6 (upper limit of normal: 7.0). After 4 weeks, LCI improved by -0.36 after AZLI and deteriorated by +0.12 after tobramycin treatment (p = 0.039). No significant differences between treatments (p = 0.195) were observed using FEV1. These results suggest that lung clearance index can be used to detect treatment induced changes in subjects with mild lung disease. [ABSTRACT FROM AUTHOR]

Aims: Glucocorticoid (GC) stimulation has been shown to increase cardiac contractility by elevated intracellular [Ca] but the sources for Ca entry are unclear. This study aims to determine the role of store-operated Ca entry (SOCE) for GC-mediated inotropy. Methods and results: Dexamethasone (Dex) pretreatment significantly increased cardiac contractile force ex vivo in Langendorff-perfused Sprague-Dawley rat hearts (2 mg/kg BW i.p. Dex 24 h prior to experiment). Moreover, Ca transient amplitude as well as fractional shortening were significantly enhanced in Fura-2-loaded isolated rat ventricular myocytes exposed to Dex (1 mg/mL Dex, 24 h). Interestingly, these Dex-dependent effects could be abolished in the presence of SOCE-inhibitors SKF-96356 (SKF, 2 μM) and BTP2 (5 μM). Ca transient kinetics (time to peak, decay time) were not affected by SOCE stimulation. Direct SOCE measurements revealed a negligible magnitude in untreated myocytes but a dramatic increase in SOCE upon Dex-pretreatment. Importantly, the Dex-dependent stimulation of SOCE could be blocked by inhibition of serum and glucocorticoid-regulated kinase 1 (SGK1) using EMD638683 (EMD, 50 μM). Dex preincubation also resulted in increased mRNA expression of proteins involved in SOCE (stromal interaction molecule 2, STIM2, and transient receptor potential cation channels 3/6, TRPC 3/6), which were also prevented in the presence of EMD. Conclusion: Short-term GC-stimulation with Dex improves cardiac contractility by a SOCE-dependent mechanism, which appears to involve increased SGK1-dependent expression of the SOCE-related proteins. Since Ca transient kinetics were unaffected, SOCE appears to influence Ca cycling more by an integrated response across multiple cardiac cycles but not on a beat-to-beat basis. [ABSTRACT FROM AUTHOR]

Objective: The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells. Methods: Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia. Results: At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 μM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect. Conclusion: GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells. [ABSTRACT FROM AUTHOR]

Functional variability among boys with Duchenne muscular dystrophy (DMD) is well recognised and complicates interpretation of clinical studies. We hypothesised that boys with DMD could be clustered into groups sharing similar trajectories of ambulatory function over time, as measured by the North Star Ambulatory Assessment (NSAA) total score. We also explored associations with other variables such as age, functional abilities, and genotype. Using the NorthStar Clinical Network database, 395 patients with >1 NSAA assessment were identified. We utilised latent class trajectory analysis of longitudinal NSAA scores, which produced evidence for at least four clusters of boys sharing similar trajectories versus age in decreasing order of clinical severity: 25% of the boys were in cluster 1 (NSAA falling to ≤ 5 at age ~10y), 35% were in cluster 2 (NSAA ≤ 5 ~12y), 21% in were cluster 3 (NSAA≤ 5 ~14y), and 19% in cluster 4 (NSAA > 5 up to 15y). Mean ages at diagnosis of DMD were similar across clusters (4.2, 3.9, 4.3, and 4.8y, respectively). However, at the first NSAA assessment, a significant (p<0.05) association was observed between earlier declining clusters and younger age, worse NSAA, slower rise from supine, slower 10 metre walk/run times, and younger age of steroid initiation. In order to assess the probability of observing complete loss of function for individual NSAA items, we examined the proportion of patients who shifted from a score of 1 or 2 at baseline to a score of 0. We also assessed the probability of gain of function using the inverse assessment and stratified the probability of deterioration, improvement–or static behavior–by age ranges and using baseline functional status. Using this tool, our study provides a comprehensive assessment of the NSAA in a large population of patients with DMD and, for the first time, describes discrete clusters of disease progression; this will be invaluable for future DMD clinical trial design and interpretation of findings. [ABSTRACT FROM AUTHOR]

Background: In Fetal Growth Restriction ‘fetal programming’ may take place via DNA methylation, which has implications for short-term and long-term health outcomes. Small-for-gestational age fetuses are considered fetal growth restricted, characterized by brain-sparing when fetal Doppler hemodynamics are abnormal, expressed as a cerebroplacental ratio (CPR) <1. We aimed to determine whether brain-sparing is associated with altered DNA methylation of selected genes. Methods: We compared DNA methylation of six genes in 41 small-for-gestational age placentas with a normal or abnormal CPR. We selected EPO, HIF1A, VEGFA, LEP, PHLDA2, and DHCR24 for their role in angiogenesis, immunomodulation, and placental and fetal growth. DNA methylation was analyzed by pyrosequencing. Results: Growth restricted fetuses with an abnormal CPR showed hypermethylation of the VEGFA gene at one CpG (VEGFA-309, p = .001) and an overall hypomethylation of the LEP gene, being significant at two CpGs (LEP-123, p = .049; LEP-51, p = .020). No differences in methylation were observed for the other genes. Conclusions: VEGFA and LEP genes are differentially methylated in placentas of small-for-gestational age fetuses with brain-sparing. Hypermethylation of VEGFA-309 in abnormal CPR-placentas could indicate successful compensatory mechanisms. Methylation of LEP-51 is known to suppress LEP expression. Hypomethylation in small-for-gestational age placentas with abnormal CPR may result in hyperleptinemia and predispose to leptin-resistance later in life. [ABSTRACT FROM AUTHOR]

Introduction: The treatment of burn wounds and hypopigmentation conditions often require autologous transplantation of keratinocytes and melanocytes. Tracking transplanted cells to ascertain their contribution to tissue recapitulation presents a challenge. This study demonstrates a methodology based on passive staining with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) that enables localization of cells in tissue sections to investigate the fate of transplanted cells in wound re-epithelialisation. Methods: Viability and migration of CFSE-stained keratinocytes and melanocytes were investigated using viability staining and scratch assays, while proliferation of cells was measured using flow cytometry. In addition, CFSE-stained keratinocytes and melanocytes were transplanted to a human ex vivo wound model, either in suspension, or with the aid of macroporous gelatine microcarriers. Wounds were analysed seven, 14 and 21 days post transplantation using cryosectioning and fluorescence microscopy. Sections from wounds with transplanted co-cultured keratinocytes and melanocytes were stained for pancytokeratin to distinguish keratinocytes. Results: CFSE-staining of keratinocytes and melanocytes did not affect the viability, migration or proliferation of the cells. Transplanted cells were tracked in ex vivo wounds for 21 days, illustrating that the staining had no effect on wound re-epithelialisation. In conclusion, this study presents a novel application of CFSE-staining for tacking transplanted primary human keratinocytes and melanocytes. [ABSTRACT FROM AUTHOR]

Foraminifera are expected to be particularly susceptible to future changes in ocean carbonate chemistry as a function of increased atmospheric CO2. Studies in an experimental recirculating seawater system were performed with a dominant benthic foraminiferal species collected from intertidal mudflats. We investigated the experimental impacts of ocean acidification on survival, growth/calcification, morphology and the biometric features of a calcareous species Elphidium williamsoni. Foraminifera were exposed for 6 weeks to four different pH treatments that replicated future scenarios of a high CO2 atmosphere resulting in lower seawater pH. Results revealed that declining seawater pH caused a decline in foraminiferal survival rate and growth/calcification (mainly through test weight reduction). Scanning electron microscopy image analysis of live specimens at the end of the experimental period show changes in foraminiferal morphology with clear signs of corrosion and cracking on the test surface, septal bridges, sutures and feeding structures of specimens exposed to the lowest pH conditions. These findings suggest that the morphological changes observed in shell feeding structures may serve to alter: (1) foraminiferal feeding efficiency and their long-term ecological competitiveness, (2) the energy transferred within the benthic food web with a subsequent shift in benthic community structures and (3) carbon cycling and total CaCO3 production, both highly significant processes in coastal waters. These experimental results open-up the possibility of modelling future impacts of ocean acidification on both calcification and dissolution in benthic foraminifera within mid-latitude intertidal environments, with potential implications for understanding the changing marine carbon cycle. [ABSTRACT FROM AUTHOR]

L-5-methyltetrahydrofolate is the predominant folate form in human milk but is currently not approved as a folate source for infant and follow-on formula. We aimed to assess the suitability of L-5-methyltetrahydrofolate as a folate source for infants. Growth and tolerance in healthy term infants fed formulae containing equimolar doses of L-5-methyltetrahydrofolate (10.4 μg/ 100 ml, n = 120, intervention group) or folic acid (10.0 μg/ 100 ml, n = 120, control group) was assessed in a randomized, double-blind, parallel, controlled trial. A reference group of breastfed infants was followed. Both formulae were well accepted without differences in tolerance or occurrence of adverse events. The most common adverse events were common cold, poor weight gain or growth, rash, eczema, or dry skin and respiratory tract infection. Weight gain (the primary outcome) was equivalent in the two groups (95% CI -2.11; 1.68 g/d). In line with this, there was only a small difference in absolute body weight adjusted for birth weight and sex at visit 4 (95% CI -235; 135 g). Equivalence was also shown for gain in head circumference but not for recumbent length gain and increase in calorie intake. Given the nature of the test, this does not indicate an actual difference, and adjusted means at visit 4 were not significantly different for any of these parameters. Infants receiving formula containing L-5-methyltetrahydrofolate had lower mean plasma levels of unmetabolized folic acid (intervention: 0.73 nmol/L, control: 1.15 nmol/L, p<0.0001) and higher levels of red cell folate (intervention: 907.0 ±192.8 nmol/L, control: 839.4 ±142.4 nmol/L, p = 0.0095). We conclude that L-5-methyltetrahydrofolate is suitable for use in infant and follow-on formula, and there are no indications of untoward effects. Trial registration: This trial was registered at ClinicalTrials.gov (). [ABSTRACT FROM AUTHOR]

Human fumarylacetoacetate hydrolase (FAH) domain containing protein 1 (FAHD1) is a mitochondrial oxalocatate decarboxylase, the first of its kind identified in eukaryotes. The physiological role of FAHD1 in other eukaryotes is still poorly understood. In C. elegans loss of the FAHD1 ortholog FAHD-1 was reported to impair mitochondrial function, locomotion and egg-laying behavior, yet the underlying mechanisms remained unclear. Using tissue-specific rescue of fahd-1(-) worms, we find that these phenotypic abnormalities are at least in part due to fahd-1’s function in neurons. Moreover, we show that egg-laying defects in fahd-1(-) worms can be fully rescued by external dopamine administration and that depletion of fahd-1 expression induces expression of several enzymes involved in serotonin biosynthesis. Together, our results support a role for fahd-1 in modulating serotonin levels and suggest this protein as a novel link between metabolism and neurotransmitter signaling in the nervous system. Finally, we propose a model to explain how a metabolic defect could ultimately lead to marked changes in neuronal signaling. [ABSTRACT FROM AUTHOR]

The energy expenditure and substrate utilisation were measured in 5 men pre- and post- a 67 day, 1750km unassisted Antarctic traverse from the Hercules Inlet to the Ross Sea Ice via the South pole pulling sledges weighing 120kg whilst experiencing temperatures as low as -57°C. A 36-hours protocol in a whole body calorimeter was employed to measure periods of rest, sleep and three periods of standardised stepping exercises at 80, 100 and 120 steps min-1; participants were fed isocalorically. Unlike previous expeditions where large weight loss was reported, only a modest loss of body weight (7%, P = 0.03) was found; fat tissue was reduced by 53% (P = 0.03) together with a small, but not statistically significant, increase in lean tissue weight (P = 0.18). This loss occurred despite a high-energy intake (6500 kcal/day) designed to match energy expenditure. An energy balance analysis suggested the loss in body weight could be due to the energy requirements of thermoregulation. Differences in energy expenditure [4.9 (0.1) vs 4.5 (0.1) kcal/min. P = 0.03], carbohydrate utilisation [450 (180) vs 569 (195) g/day; P = 0.03] and lipid utilisation [450 (61) vs 388 (127) g/day, P = 0.03] at low levels of exertion were different from pre-expedition values. Only carbohydrate utilisation remained statistically significant when normalised to body weight. The differences in energy expenditure and substrate utilisation between the pre- and post-expedition for other physiological states (sleeping, resting, higher levels of exercise, etc) were small and not statistically significant. Whilst inter-subject variability was large, there was a tendency for increased carbohydrate utilisation, post-expedition, when fasted that decreased upon feeding. [ABSTRACT FROM AUTHOR]

During the lactation period, rat pups are fed by the dam, and the patterns of mother-pup interaction change during this period. Additionally, there are changes in feeding; first, mother´s milk is the only food needed for sustenance, and later, it is combined with solid food and water. GH serum concentrations depend on both maternal-pup interaction and energy metabolism. In the artificial rearing (AR) procedure, pups are deprived of mother-pup interaction, and the feeding pattern is controlled. This rearing paradigm has been used in rats to analyze the effects of maternal deprivation on social behavior. In the present study, we analyzed the variation in GH, acylated ghrelin and IGF-1 serum concentrations throughout the lactation period in AR pups. At pnd7, the maternal rearing (MR) pups responded to a 4 h fast with a drop in GH serum concentration, which is a well-known response to maternal deprivation. GH serum levels in the AR pups did not change, suggesting an adaptation phenomenon. A dopamine inhibitory effect of GH secretion was observed in pnd7 cultured somatotropes, suggesting dopamine regulation of GH secretion at this age. Acylated ghrelin serum levels in the AR pups showed an inverted pattern compared to that in the MR pups, which was related to the artificial feeding pattern. IGF-1 serum levels were lower in the AR pups than in MR pups, which was associated with hepatic GH resistance and with low Igf1 mRNA expression at pnd7. Interestingly, at pnd14, both pup groups showed high hepatic Igf1 mRNA expression but low IGF-1 serum levels, and this was inverted at pnd21. However, serum glucose levels were lower in the AR pups at pnd14 but reached the same levels as the MR pups at pnd21. Moreover, hepatomegaly and higher hepatic GH-receptor levels were observed in the AR pups at pnd21, which was in agreement with an absence of a solid food meal. During AR, the pups lost the maternal interaction-stimulated GH secretion, which correlated with lower IGF-1 serum levels during the first week of postnatal development. Later, the AR pups exhibited hepatic responses, in order to satisfy the metabolic demand for the normal weaning, with low carbohydrates levels in their meal. [ABSTRACT FROM AUTHOR]

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse event of bone-modifying agents used to prevent bone complications in cancer patients with bone metastasis. Currently, early treatment is the only way to prevent further progression, as the pathogenesis of MRONJ has not yet been elucidated, and a standard treatment has not been established. The aim of this study was to identify a marker for early detection marker of MRONJ by exploring substances in saliva specific to MRONJ at an early stage. We collected salivary samples from 17 patients with MRONJ and conducted metabolomic analyses using capillary electrophoresis mass spectrometry for non-targeted analysis of hydrophilic metabolites. In the screening cohort, we compared the saliva of patients with stage ≥1 advanced MRONJ (n = 9) with that of controls without MRONJ before chemotherapy (n = 9). The top 5 most elevated salivary metabolites were histamine, 3-(4-hydroxyphenyl)propionate, malonate, carnosine, and hypotaurine. In the validation cohort, we analyzed additional patients with stage ≥1 advanced MRONJ (n = 8) and controls without MRONJ after chemotherapy (n = 9), confirming a significant 2.28-fold elevation in the salivary concentration of hypotaurine. These results revealed elevated salivary hypotaurine concentration as a potential marker for the early detection of MRONJ. [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]

Coccolith mass is an important parameter for estimating coccolithophore contribution to carbonate sedimentation, organic carbon ballasting and coccolithophore calcification. Single coccolith mass is often estimated based on the ks model, which assumes that length and thickness increase proportionally. To evaluate this assumption, this study compared coccolith length, thickness, and mass of seven Emiliania huxleyi strains and one Gephyrocapsa oceanica strain grown in 25, 34, and 44 salinity artificial seawater. While coccolith length increased with salinity in four E. huxleyi strains, thickness did not increase significantly with salinity in three of these strains. Only G. oceanica showed a consistent increase in length with salinity that was accompanied by an increase in thickness. Coccolith length and thickness was also not correlated in 14 of 24 individual experiments, and in the experiments in which there was a positive relationship r2 was low (<0.4). Because thickness did not increase with length in E. huxleyi, the increase in mass was less than expected from the ks model, and thus, mass can not be accurately estimated from coccolith length alone. [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]

Although the literature on the Maillard reaction in infant formulas is extensive, most studies have focused on model systems, and in only a few cases on real food systems. Therefore, the objective of the present study was to determine the status of the Maillard reaction, both the early and advanced phases, in a variety of commercial infant formulas available on the Swedish market. Ten powder and liquid milk-based infant formulas from three manufacturers were selected to determine available lysine and CML contents, the two established indicators of the reaction. The products were also characterized with respect to protein content, carbohydrates composition, water content and water activity. In order to be able to compare the impact of different processing steps applied on powder and liquid formulas, the solid formulas contained similar ingredients as their corresponding liquid ones. Our findings showed that powder and liquid formulas contained similar available lysine concentrations regardless of the manufacturer, showing 27.14–36.57% decrease in the available lysine, compared to the reference skim milk powder in this study. The CML concentrations were in a broad range of 68.77–507.99 mg / kg protein. In the case of one manufacturer, liquid infant formulas had significantly higher CML content, compared to the powder products (p < 0.05). The results from this study are a step taken towards better understanding of the extent of the Maillard reaction in real complex systems of infant formulas. [ABSTRACT FROM AUTHOR]

In 2017, the WHO identified Acinetobacter baumannii as the top priority for the development of new antibiotics. Despite the need for new antibiotics, there remains a lack of well validated preclinical tools for A. baumannii. Here, we characterize and validate a mouse model for A. baumannii translational research. Antibiotic sensitivity for meropenem, amikacin, and polymyxin b was determined by the broth microdilution MIC assay. LD100 inoculums, in both blood and lung infection models, were determined in male and female C3HeB/FeJ mice that were challenged with various A. baumannii clinical isolates. Blood (blood infection model) or blood and lung tissue (lung infection model) were collected from infected mice at 2 and 18 hours and the bacterial burden was determined by quantitative culture. Blood chemistry was analyzed using the iStat system. Cytokines (IL-1ß, TNF, IL-6, and IL-10) were measured in the blood and lung homogenate by ELISA assay. Lung sections (H&E stains) were scored by a pathologist. In the blood infection model, the cytokines and physiological data indicate that mice become moribund due to sepsis (low blood pH, falling bicarbonate, and a rising base deficit), whereas mice become moribund due to respiratory failure (low blood pH, rising bicarbonate, and a falling base deficit) in the oral aspiration pneumonia model. We also characterized the timing of changes in various clinical and biomarker endpoints, which can serve as a basis for future interventional studies. Susceptibility was generally similar across gender and infection route. However, we did observe that female mice were approximately 2-fold more sensitive to LAC-4 ColR in the blood infection model. We also observed that female mice were more than 10-fold more resistant to VA-AB41 in the oral aspiration pneumonia model. These results establish parameters to follow in order to assess efficacy of novel preventative and therapeutic approaches for these infections. [ABSTRACT FROM AUTHOR]

Glycoconjugation to target the Warburg effect provides the potential to enhance selective uptake of anticancer or imaging agents by cancer cells. A Warburg effect targeting group, rationally designed to facilitate uptake by glucose transporters and promote cellular accumulation due to phosphorylation by hexokinase (HK), has been synthesised. This targeting group, the C2 modified glucose analogue 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-D-glucose, has been conjugated to the fluorophore nitrobenzoxadiazole to evaluate its effect on uptake and accumulation in cancer cells. The targeting vector has demonstrated inhibition of glucose phosphorylation by HK, indicating its interaction with the enzyme and thereby confirming the potential to facilitate an intracellular trapping mechanism for compounds it is conjugated with. The cellular uptake of the fluorescent analogue is dependent on the glucose concentration and is so to a greater extent than is that of the widely used fluorescent glucose analogue, 2-NBDG. It also demonstrates selective uptake in the hypoxic regions of 3D spheroid tumour models whereas 2-NBDG is distributed primarily through the normoxic regions of the spheroid. The increased selectivity is consistent with the blocking of alternative uptake pathways. [ABSTRACT FROM AUTHOR]

The biopharmaceutical industry is growing at a fast pace, making nowadays 20% of the pharma market. Within this market, therapeutic monoclonal antibodies (mAbs) are the dominant product class. With the patent expirations, biosimilars and, perhaps more relevant, biobetters, are in fast development. Thus, a comprehensive characterization at the molecular level of antibodies heterogeneity such as glycoforms, post-translational modifications (PTMs) and sequence variations is of utmost importance. Mass spectrometry (MS)-based approaches are undoubtedly the most powerful analytical strategies to monitor and define an array of critical quality attributes on mAbs. In this work, we demonstrate the analytical power of the Q-TOF MS platform for comprehensive and detailed analysis at molecular levels of an in-house produced mAb. This methodology involves minimal sample preparation procedures and provides an extensive collection of valuable data in a short period of time. [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]

Despite having been sequenced over a decade ago, the functional significance of much of the mammalian genome remains unknown. The mouse has become the preeminent mammalian model for identifying endogenous gene function in vivo. Here we characterize the phenotype of a loss-of function allele for the evolutionarily conserved transcription factor, Elongation Factor Homolog 1 (Elof1). Recent work utilizing the yeast homolog, Elf1, has demonstrated that Elf1 associates with the RNA polymerase II complex to promote elongation by relieving the association of the template DNA strand with bound histones. Loss of Elof1 results in developmental delay and morphological defects during early mouse development resulting in peri-gastrulation lethality. Although Elof1 is highly conserved we observe tissue specific expression during gastrulation and in adult murine tissues, suggesting there may be other genes with similar function in diverse tissues or that mElof1 has adopted lineage specific functions. To better understand its function in mammalian transcription, we examined splice variants and find that Elof1 regulates mutually exclusive exon use in vivo. Distinct from what has been demonstrated in yeast, we demonstrate that Elof1 is essential for viability during mammalian gastrulation which may be due to a role mediating tissue specific exclusive exon use, a regulatory function unique to higher eukaryotes. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations in the gene encoding the epithelial chloride channel CF transmembrane conductance regulator (CFTR) protein. The most common mutation is a deletion of three nucleotides leading to the loss of phenylalanine at position 508 (p.Phe508del) in the protein. This study evaluates eluforsen, a novel, single-stranded, 33-nucleotide antisense oligonucleotide designed to restore CFTR function, in in vitro and in vivo models of p.Phe508del CF. The aims of the study were to demonstrate cellular uptake of eluforsen, and its efficacy in functional restoration of p.Phe508del-CFTR both in vitro and in vivo. In vitro, the effect of eluforsen was investigated in human CF pancreatic adenocarcinoma cells and human bronchial epithelial cells. Two mouse models were used to evaluate eluforsen in vivo. In vitro, eluforsen improved chloride efflux in CF pancreatic adenocarcinoma cell cultures and increased short-circuit current in primary human bronchial epithelial cells, both indicating restoration of CFTR function. In vivo, eluforsen was taken up by airway epithelium following oro-tracheal administration in mice, resulting in systemic exposure of eluforsen. In female F508del-CFTR mice, eluforsen significantly increased CFTR-mediated saliva secretion (used as a measure of CFTR function, equivalent to the sweat test in humans). Similarly, intranasal administration of eluforsen significantly improved nasal potential difference (NPD), and therefore CFTR conductance, in two CF mouse models. These findings indicate that eluforsen improved CFTR function in cell and animal models of p.Phe508del-CFTR-mediated CF and supported further development of eluforsen in human clinical trials, where eluforsen has also been shown to improve CFTR activity as measured by NPD. [ABSTRACT FROM AUTHOR]

The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT. [ABSTRACT FROM AUTHOR]

Absence labels promote the absence of a particular ingredient or production practice. Consumers usually perceive organic labels as an umbrella absence label for a variety of ingredients and production practices. Such organic labels often use similar language but are based on different certification requirements. For example, both organic wine and wine made with organic grapes are available to U.S. consumers, but little is known about consumer preferences for such labeled products when information about the certification standards is available. Moreover, while absence labels, which advertise the absence of certain attributes or practices, are prevalent on the market, little is known about how information on conventional production practices impacts consumer behavior. Using an artefactual experiment with 128 adult non-student participants, we investigate consumer demand for conventional wine, organic wine, and wine made with organic grapes when information about production standards is provided to participants with and without details regarding conventional winemaking practices. We find that while both organic labels carry a significant and very similar willingness-to-pay (WTP) premium, information about certification standards and conventional wine making practices can reduce WTP for all wines. Providing information about the two organic certification standards reduces consumer WTP for both absence labeled and conventional wine categories. This effect largely disappears for organic wine, but not wine made with organic grapes, when information about conventional wine-making practices is also provided. [ABSTRACT FROM AUTHOR]

In insects, the gustatory system has a critical function not only in selecting food and feeding behaviours but also in growth and metabolism. Gustatory receptors play an irreplaceable role in insect gustatory signalling. Trichogramma chilonis is an effective biocontrol agent against agricultural insect pests. However, the molecular mechanism of gustation in T. chilonis remains elusive. In this study, we found that T. chilonis adults had a preference for D-fructose and that D-fructose contributed to prolong longevity and improve fecundity. Then, We also isolated the full-length cDNA encoding candidate gustatory receptor (TchiGR43a) based on the transcriptome data of T. chilonis, and observed that the candidate gustatory receptor gene was expressed from the larval to adult stages. The expression levels of TchiGR43a were similar between female and male. A Xenopus oocyte expression system and two-electrode voltage-clamp recording further verified the function analysis of TchiGR43a. Electrophysiological results showed that TchiGR43a was exclusively tuned to D-fructose. By the studies of behaviour, molecular biology and electrophysiology in T. chilonis, our results lay a basic fundation of further study on the molecular mechanisms of gustatory reception and provide theoretical basis for the nutritional requirement of T. chilonis in biocontrol. [ABSTRACT FROM AUTHOR]

The signaling lymphocytic activation molecule (SLAM) family is comprised of nine distinct receptors that are expressed exclusively on hematopoietic cells. Most of these transmembrane receptors are homotypic by nature and downstream signaling occurs when cells that express the same SLAM receptor interact. Previous studies have determined that anti-SLAMF6 antibodies can have a therapeutic effect in autoimmunity and cancer. However, little is known about the role of SLAMF6 in the adaptive immune responses and in order to utilize SLAMF6 interventional approaches, a better understanding of the biology of this receptor in T cell is warranted. Accordingly, the objective of our study was to investigate both functionally and structurally the role of SLAMF6 in T cell receptor (TCR) mediated responses. Biochemical and genetic experiments revealed that SLAMF6 was required for productive TCR downstream signaling. Interestingly, SLAMF6 ectodomain was required for its function, but not for its recruitment to the immunological synapse. Flow-cytometry analysis demonstrated that tyrosine 308 of the tail of SLAMF6 was crucial for its ability to enhance T cell function. Imaging studies revealed that SLAMF6 clustering, specifically with the TCR, resulted in dramatic increase in downstream signaling. Mechanistically, we showed that SLAMF6 enhanced T cell function by increasing T cell adhesiveness through activation of the small GTPase Rap1. Taken together SLAMF6 is an important regulator of T cell activation where both its ectodomain and its endodomain contribute differentially to T cell functions. Additional studies are underway to better evaluate the role of anti-SLAMF6 approaches in specific human diseases. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase receptor type Z (PTPRZ) is preferentially expressed in the central nervous system as two transmembrane receptor isoforms PTPRZ-A/B and one secretory isoform PTPRZ-S. Ptprz-knockout mice lacking the expression of all three isoforms show behavioral, learning, and neurological abnormalities, including increased exploratory activities to novelty, deficits in spatial and contextual learning, and reduced responses to methamphetamine, relative to wild-type mice. To investigate whether PTPRZ isoforms play distinct physiological roles, we herein performed behavioral studies on two knock-in mouse lines: One expresses the catalytically inactive Cys-1930 to Ser (CS) mutants of PTPRZ-A/B, while the other generated in the present study expresses catalytically active mutants of PTPRZ-A/B lacking the negative regulatory PTP-D2 domain and C-terminal PDZ-binding motif (ΔD2) instead of wild-type PTPRZ-A/-B. In contrast to Ptprz-knockout mice, neither increased responses to novelty in the open field nor memory impairments in the inhibitory-avoidance task were observed in Ptprz-CS or Ptprz-ΔD2 mice. However, the effects of methamphetamine on locomotor activity were significantly weaker in Ptprz-KO mice and CS mutant mice than in wild-type mice, but were normal in ΔD2 mutant mice. Furthermore, microdialysis experiments revealed that methamphetamine-evoked dopamine release in the nucleus accumbens was reduced in Ptprz-KO mice and CS mutant mice. These results suggest that the extracellular region of PTPRZ, including the secretory isoform, is crucial for behavioral responses to novelty and the formation of aversive memories, whereas the PTPase activities of PTPRZ receptor isoforms are involved in regulating the dopaminergic system. [ABSTRACT FROM AUTHOR]

Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) is a promising quite new polymer with very interesting properties. The thermal degradation process of PDMAEMA was investigated. The polymer was heated at specific time intervals, then heating was stopped, and infrared analysis was performed to obtain information on the structure of the solid residue. The thermal degradation process has a two-stage character. The limit temperature for the first decomposition step was about 390°C, after which the second stage of sample decomposition began. The order of disintegration of the macromolecules was determined. Activation energy values for the thermal decomposition process have been calculated; they are 89.8 kJ/mol for the first stage and 17.7 kJ/mol for the second stage of the degradation process. [ABSTRACT FROM AUTHOR]

Introduction: Despite advances in perioperative management and surgical technique, postoperative liver failure remains a feared complication after hepatic resection. Various supportive treatment options are under current discussion, but lack of structured evaluation. We therefore established a porcine model of major liver resection to study regeneration after partial hepatectomy in a reliable and well-defined pre-clinical setting. Methods: Major hepatectomy was performed on seven minipigs with the intention to set up a non-lethal but relevant transient impairment of liver function. For steady postoperative vascular access (e.g. for blood withdrawal, measurement of venous pressure), permanent catheters were implanted into the internal jugular and portal veins, respectively. Animals were followed up for 30 days; clinical and laboratory results were recorded in detail. Monitoring was enhanced by non-invasive determination of the maximum liver function capacity (LiMAx test). Results and conclusions: The established porcine model appeared suitable for evaluation of postoperative liver regeneration. Clinical characteristics and progression of liver function impairment as well as subsequent recovery were comparable to courses known from surgery in humans. Laboratory parameters (e.g. liver enzymes, bilirubin, INR, coagulation factor II) showed relevant derangements during postoperative days (POD) 0 to 3 followed by normalization until POD 7. Application of the LiMAx test was feasible in minipigs, again showing values comparable to humans and kinetics in line with obtained laboratory parameters. The exteriorized portal vein catheters enabled intra- and postoperative monitoring of portal venous pressures as well as easy access for blood withdrawal without relevant risk of postoperative complications. [ABSTRACT FROM AUTHOR]

Background/Aim: Patients with polycystic ovary syndrome (PCOS), characterized by anovulation, hyperandrogenemia and polycystic ovaries, are still vulnerable to undergo recurrent pregnancy loss and premature labor even though the ovulatory process is pharmacologically recovered. However, its potential mechanism remains unknown. Thus, our aim was to investigate the effect and mechanism of hyperandrogenemia and flutamide (a non-steroidal anti-androgen) on the embryo implantation and pregnancy during mid-pregnancy. Methods: We used a mouse model in which PCOS-like hyperandrogenemia was induced by subcutaneous injection of testosterone propionate. In this model, we observed the effect of hyperandrogenemia and flutamide on the decidualization, angiogenesis and uNK cells by methods of immunohistochemistry, quantitative PCR, western blotting and Dolichos biflorus agglutinin (DBA) lectin staining. Results: Testosterone and flutamide treatment did not significantly influence the numbers of implanted embryo compared with the control group. However, different doses of testosterone significantly increased the ratio of resorbed /implanted embryo, decreased the level of prl8a2 mRNA and cyclin D3 protein, inhibited the uterine angiogenesis and reduced the numbers of uNK cells, but combined treatment with flutamide markedly decreased the resorbed embryos, increased expressions of prl8a2 mRNA and cyclin D3 protein and angiogenesis and numbers of uNK cells. Conclusion: Flutamide treatment can efficiently ameliorate the hyperandrogenemia-induced the disorders in aspects of decidualization, angiogenesis and uNK cells, which further improve the poor endometrial receptivity in PCOS patients. [ABSTRACT FROM AUTHOR]

Purpose: To assess the physiology of the retina by electroretinography (ERG) with skin electrodes in eyes that had undergone vitrectomy with silicone oil (SO) tamponade. Design: Retrospective case series. Method: ERGs were recorded from eleven eyes with complex vitreoretinal disorders and from the normal fellow eyes. The affected eyes underwent pars plana vitrectomy (PPV) with SO tamponade. ERGs were recorded before and after the SO was removed. The amplitudes and implicit times of the a- and b-waves of the affected eyes were compared to those of the normal fellow eyes. In addition, the ratios of the amplitudes of the b-waves of the affected eyes to those of the fellow eyes were compared before and after the SO was removed. Results: ERGs were recordable from 7 eyes (63.6%) before the SO was removed and 11 eyes (100%) after the SO was removed. The a- and b-wave amplitudes were significantly smaller in the affected eyes than those of the fellow eyes at the baseline. The b-wave amplitude before the removal of the SO was significantly and positively correlated with that after the SO removal. The ratios of the b-waves of the affected/normal fellow eye significantly increased after the SO was removed. Conclusion: The results indicate that ERGs picked up by skin electrode can be used to assess the physiology of the retina in eyes with a SO tamponade. The amplitude of the b-waves of the ERGs in silicone-filled eyes can be used to predict the amplitude after the silicone is removed. [ABSTRACT FROM AUTHOR]

Mannitol, a sugar alcohol used in commercial food products, has been previously shown to induce sex-biased mortality in female Drosophila melanogaster when ingested at a single concentration (1 M). We hypothesized that sex differences in energy needs, related to reproductive costs, contributed to the increased mortality we observed in females compared to males. To test this, we compared the longevity of actively mating and non-mating flies fed increasing concentrations of mannitol. We also asked whether mannitol-induced mortality was concentration-dependent for both males and females, and if mannitol’s sex-biased effects were consistent across concentrations. Females and males both showed concentration-dependent increases in mortality, but female mortality was consistently higher at concentrations of 0.75 M and above. Additionally, fly longevity decreased further for both sexes when housed in mixed sex vials as compared to single sex vials. This suggests that the increased energetic demands of mating and reproduction for both sexes increased the ingestion of mannitol. Finally, larvae raised on mannitol produced expected adult sex ratios, suggesting that sex-biased mortality due to the ingestion of mannitol occurs only in adults. We conclude that sex and reproductive status differences in mannitol ingestion drive sex-biased differences in adult fly mortality. [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]