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An ongoing loss of experts in marine cellular biochemistry and physiology (CBP) is stagnating the generation of knowledge upon which rapidly growing "omics" approaches rely, ultimately hampering our ability to predict organismal responses to climate change. The ongoing loss of expertise on the biochemistry and physiology of marine organisms is hampering our understanding of biological mechanisms and will ultimately affect our ability to predict organismal responses to climate change. [ABSTRACT FROM AUTHOR]

Background: Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue. We now aim to determine the impact of hypoxia on islet vascularization. Methods: We induced hypoxia in cultured mouse islets using the hypoxia mimetic cobalt chloride (100 μM CoCl2). We measured the impact on islet metabolism (two-photon NAD(P)H and Rh123 imaging) and function (insulin secretion and survival). We also measured the impact on hypoxia related transcripts (HIF-1α, VEGF-A, PDK-1, LDHA, COX4) and confirmed increased VEGF-A expression and secretion. Finally, we measured the vascularization of islets in static and flowing culture using PECAM-1 immunofluorescence. Results: CoCl2 did not induce significant changes in beta cell metabolism (NAD(P)H and Rh123), insulin secretion, and survival. Consistent with hypoxia induction, CoCl2 stimulated HIF-1α, PDK-1, and LDHA transcripts and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2) and this switch was noted in the glucose-stimulated cytoplasmic NAD(P)H responses. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static (dish) and microfluidic flow culture. Conclusions: Hypoxia induction using CoCl2 had a positive effect on islet EC morphology and survival with limited impact on beta-cell metabolism, function, and survival. The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin. [ABSTRACT FROM AUTHOR]

Introduction: While extracorporeal membrane oxygenation (ECMO) is effective in preventing further hypoxemia and maintains blood flow in endotoxin-induced shock, ECMO alone does not reverse the hypotension. In this study, we tested whether concurrent vasopressor use with ECMO would provide increased circulatory support and blood flow, and characterized regional blood flow distribution to vital organs. Methods: Endotoxic shock was induced in piglets to achieve a 30% decrease in mean arterial pressure (MAP). Measurements of untreated pigs were compared to pigs treated with ECMO alone or ECMO and vasopressors. Results: ECMO provided cardiac support during vasodilatory endotoxic shock and improved oxygen delivery, but vasopressor therapy was required to return MAP to normotensive levels. Increased blood pressure with vasopressors did not alter oxygen consumption or extraction compared to ECMO alone. Regional microcirculatory blood flow (RBF) to the brain, kidney, and liver were maintained or increased during ECMO with and without vasopressors. Conclusion: ECMO support and concurrent vasopressor use improve regional blood flow and oxygen delivery even in the absence of full blood pressure restoration. Vasopressor-induced selective distribution of blood flow to vital organs is retained when vasopressors are administered with ECMO. [ABSTRACT FROM AUTHOR]

Early clinical successes are driving enthusiasm for fecal microbiota transplantation (FMT), the transfer of healthy gut bacteria through whole stool, as emerging research is linking the microbiome to many different diseases. However, preliminary trials have yielded mixed results and suggest that heterogeneity in donor stool may play a role in patient response. Thus, clinical trials may fail because an ineffective donor was chosen rather than because FMT is not appropriate for the indication. Here, we describe a conceptual framework to guide rational donor selection to increase the likelihood that FMT clinical trials will succeed. We argue that the mechanism by which the microbiome is hypothesized to be associated with a given indication should inform how healthy donors are selected for FMT trials, categorizing these mechanisms into four disease models and presenting associated donor selection strategies. We next walk through examples based on previously published FMT trials and ongoing investigations to illustrate how donor selection might occur in practice. Finally, we show that typical FMT trials are not powered to discover individual taxa mediating patient responses, suggesting that clinicians should develop targeted hypotheses for retrospective analyses and design their clinical trials accordingly. Moving forward, developing and applying novel clinical trial design methodologies like rational donor selection will be necessary to ensure that FMT successfully translates into clinical impact. [ABSTRACT FROM AUTHOR]

Cells damaged by mechanical or infectious injury release proinflammatory mitochondrial DNA (mtDNA) fragments into the circulation. We evaluated the relation between plasma levels of mtDNA fragments in obese type 2 diabetes mellitus (T2DM) patients and measures of chronic inflammation and insulin resistance. In 10 obese T2DM patients and 12 healthy control (HC) subjects, we measured levels of plasma cell-free mtDNA with quantitative real-time polymerase chain reaction, and mtDNA damage in skeletal muscle with quantitative alkaline Southern blot. Also, markers of systemic inflammation and oxidative stress in skeletal muscle were measured. Plasma levels of mtDNA fragments, mtDNA damage in skeletal muscle and plasma tumor necrosis factor α levels were greater in obese T2DM patients than HC subjects. Also, the abundance of plasma mtDNA fragments in obese T2DM patients levels positively correlated with insulin resistance. To the best of our knowledge, this is the first published evidence that elevated level of plasma mtDNA fragments is associated with mtDNA damage and oxidative stress in skeletal muscle and correlates with insulin resistance in obese T2DM patients. Plasma mtDNA may be a useful biomarker for predicting and monitoring insulin resistance in obese patients. [ABSTRACT FROM AUTHOR]

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]

Background: In patients with left heart failure, micro-RNAs (miRNAs) have been shown to be of diagnostic and prognostic value. The present study aims to identify those miRNAs in patients with univentricular heart (UVH) disease that may be associated with overt heart failure. Methods: A large panel of human miRNA arrays were used to determine miRNA expression profiles in the blood of 48 UVH patients and 32 healthy controls. For further selection, the most abundantly expressed miRNA arrays were related to clinical measures of heart failure and selected miRNAs validated by polymerase chain reaction were used for the prediction of overt heart failure and all-cause mortality. Results: According to microarray analysis, 50 miRNAs were found to be significantly abundant in UVH patients of which miR-150-5p was best related to heart failure parameters. According to ROC analysis, NT-proBNP levels (AUC 0.940, 95% CI 0.873–1.000; p = 0.001), miR-150-5p (AUC 0.905, 95% CI 0.779–1.000; p = 0.001) and a higher NYHA class ≥ III (AUC 0.893, 95% CI 0.713–1.000; p = 0.002) were the 3 most significant predictors of overt heart failure. Using a combined biomarker model, AUC increased to 0.980 indicating an additive value of miR-150-5p. Moreover, in the multivariate analysis, a higher NYHA class ≥ III (p = 0.005) and miR-150-5p (p = 0.006) turned out to be independent predictors of overt heart failure. Conclusion: In patients with UVH, miR-150-5p is an independent predictor of overt heart failure and thus may be used in the risk assessment of these patients. [ABSTRACT FROM AUTHOR]

The objectives of this study were: 1) to classify animals into groups of high and low feed efficiency using two feed efficiency indexes (Residual feed intake (RFI) and residual feed intake and body weight gain (RIG)), and 2) to evaluate if pre-weaning heifer calves divergent for feed efficiency indexes exhibit differences in performance, body measurements, digestibility, energy partitioning, and nitrogen partitioning. A total of 32 Gyr heifer calves were enrolled in a 63-d trial and classified into two feed efficiency (FE) groups based on RFI and RIG (mean ± 0.5 SD). The groups were classified as high efficiency (HE) RFI (HE RFI, n = 9; HE RIG, n = 10), and low efficiency (LE) RFI (LE RFI, n = 10; LE RIG, n = 11). The remaining animals were classified as intermediate (n = 13 (RFI) and n = 11 (RIG)). HE and LE calves had RFI values of—0.052 and 0.049 kg/d (P < 0.05), respectively. The HE RFI group consumed 8.9% less solid diet than the LE RFI group. HE RFI animals exhibited an increased digestibility of crude protein and ether extract and tended to have greater total dry and organic matter digestibility. LE RFI animals had greater gross energy and nitrogen intake, though greater fecal losses resulted in a tendency to reduce energy and nitrogen use efficiency. HE and LE calves had RIG values of 0.080 and -0.077kg/d (P ≤ 0.01), respectively. HE RIG animals exhibited greater average daily gain (9.4%), body weight (BW), and heart girth, though HE RIG group exhibited narrower hip width. HE RIG animals tended to have greater ether extract digestibility but greater methane losses (% of gross energy). HE RFI in pre-weaning heifers seems to be related to differences in digestibility. Divergent animals for RIG during the assessed phase appear to differ in body measurements, which may be related to differences in the composition of the gain. [ABSTRACT FROM AUTHOR]

Several models for the long-term development of T2DM already exist, focusing on the dynamics of the interaction between glycemia, insulinemia and β-cell mass. Current models consider representative (fasting or daily average) glycemia and insulinemia as characterizing the compensation state of the subject at some instant in slow time. This implies that only these representative levels can be followed through time and that the role of fast glycemic oscillations is neglected. An improved model (DPM15) for the long-term progression of T2DM is proposed, introducing separate peripheral and hepatic (liver and kidney) insulin actions. The DPM15 model no longer uses near-equilibrium approximation to separate fast and slow time scales, but rather describes, at each step in slow time, a complete day in the life of the virtual subject in fast time. The model can thus represent both fasting and postprandial glycemic levels and describe the effect of interventions acting on insulin-enhanced tissue glucose disposal or on insulin-inhibited hepatic glucose output, as well as on insulin secretion and β-cell replicating ability. The model can simulate long-term variations of commonly used clinical indices (HOMA-B, HOMA-IR, insulinogenic index) as well as of Oral Glucose Tolerance or Euglycemic Hyperinsulinemic Clamp test results. The model has been calibrated against observational data from the Diabetes Prevention Program study: it shows good adaptation to observations as a function of very plausible values of the parameters describing the effect of such interventions as Placebo, Intensive LifeStyle and Metformin administration. [ABSTRACT FROM AUTHOR]

Swim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. However, the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms that the upper fraction of bull sperm has not only improved motility compared to the cells in the lower fraction but also shows higher metabolic rates and longer flagella. This pattern was consistent across media of two different levels of viscosity. We conclude that the motility-based separation of the swim-up technique is also reflected in underlying metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality. [ABSTRACT FROM AUTHOR]

Hypoxia and angiogenesis are critical components in the progression of solid cancer, including gastric cancers (GCs). miR-382 has been identified as a hypoxia-induced miR (hypoxamiR), but the clinical significance in GCs has not been identified yet. To explore the clinical and prognostic importance of miR-382 in GCs, the surgical specimens of 398 patients with GCs in KNU hospital in Korea, the total of 183 patients was randomly selected using simple sampling methods and big data with 446 GCs and 45 normal tissues from the data portal () were analysed. Expression of miR-382 as well as miR-210, as a positive control hypoxamiR by qRT-PCR in histologically malignant region of GCs showed significantly positive correlation (R = 0.516, p<0.001). High miR-210 and miR-382 expression was significantly correlated with unfavorable prognosis including advanced GCs (AGC), higher T category, N category, pathologic TNM stage, lymphovascular invasion, venous invasion, and perinueral invasion, respectively (all p<0.05). In univariate analysis, high miR-210 expression was significantly associated with worse overall survival (OS) (p = 0.036) but not high miR-382. In paired 60 gastric normal and cancer tissues, miR-382 expression in cancer tissues was significantly higher than normal counterpart (p = 0.003), but not miR-210 expression. However, by increasing the patient number from the big data analysis, miR-210 as well as miR-382 expression in tumor tissues was significantly higher than the normal tissues. Our results suggest that miR-382, as novel hypoxamiR, can be a prognostic marker for advanced GCs and might be correlated with metastatic potential. miR-382 might play important roles in the aggressiveness, progression and prognosis of GCs. In addition, miR-382 give a predictive marker for progression of GCs compared to the normal or preneoplastic lesion. [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]

Cell-free DNA (cfDNA) in the human blood circulation has been under investigation since its initial observation in 1948. Plasma cfDNA is known to be significantly elevated in diseased people. Due to possible variation in the population, evaluating cfDNA as a non-invasive biomarker at disease onset alone may not be sensitive enough to accurately diagnose diseases, particularly early stage cancers on a personal level. To understand the factors that define the cfDNA levels on the personal level and for better use as a non-invasive biomarker, we isolated cfDNA from the plasma of healthy individuals with varying degrees of genetic and/or environmental similarities (monozygotic twins, dizygotic twins, sibling pairs, and unrelated individuals) as well as from patients with varying stages of breast and ovarian cancer undergoing treatment. Cell-free DNA levels were quantified by a fluorometer (ng/ml) and/or real-time PCR (copies/ml). The associations between individuals with various degrees of genetic and/or environmental similarities and their plasma cfDNA levels were evaluated. The ACE model (A = additive genetic, C = common environment, and E = specific environmental factors) was used to determine the proportion of each factor on the cfDNA levels. We found a high correlation (r = 0.77; p < 0.0001) in plasma cfDNA levels between monozygotic twins (n = 39). However, the correlation was gradually reduced to moderate (r = 0.47; p = 0.016) between dizygotic twins (n = 13) and low correlation (r = 0.28; p = 0.043) between sibling pairs (n = 26). The ACE model analysis showed that the plasma cfDNA level of a given healthy individual is influenced both by genetic and the environmental components in similar proportions (53% and 47%, respectively; A = 53%, C = 22.5%, E = 24.5%). Moreover, while age had no effect, gender significantly influenced the individual's plasma cfDNA level. As expected, cfDNA levels were significantly higher in both breast (n = 26) (p<0.0001) and ovarian (n = 64) (p<0.0001) cancer patients compared to the healthy individuals. Our study demonstrated that both genome and environmental factors modulate the individual's cfDNA level suggesting that its diagnostic sensitivity may be improved only if the person's cfDNA level is known prior to disease presentation. [ABSTRACT FROM AUTHOR]

Activated platelets release adenosine trisphosphate (ATP) and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants. However, bioluminescence analysis of ATP possesses poor linearity, the signal is quickly attenuated, and the accuracy of ATP release from platelets is hard to determine accurately enough to be used in a high throughput screening of platelet inhibitors. The present study was designed to optimize bioluminescence analysis of ATP released by platelets and expand its application in high throughput screening of platelet inhibitors. The results showed that accuracy of ATP analysis was significantly improved by adding coenzyme A (CoA) and signal attenuation of ATP analysis was greatly postponed by adding bovine serum albumin (BSA) both in Hank's balanced salt solution (HBSS) and Tyrode's buffer. Furthermore, ATP release of activated platelets and inhibitory effects of Ly294002 and Staurosporine on platelet activation were accurately determined by our optimized bioluminescence analysis of ATP. Thus, we have successfully constructed an optimized bioluminescence analysis of ATP which can be used in high throughput screening of platelet inhibitors. [ABSTRACT FROM AUTHOR]

Peripheral Blood gene expression is widely used in the discovery of biomarkers and development of therapeutics. Recently, a spate of commercial blood collection and preservation systems have been introduced with proprietary variations that may differentially impact the transcriptomic profiles. Comparative analysis of these collection platforms will help optimize protocols to detect, identify, and reproducibly validate true biological variance among subjects. In the current study, we tested two recently introduced whole blood collection methods, RNAgard® and PAXgene® RNA, in addition to the traditional method of peripheral blood mononuclear cells (PBMCs) separated from whole blood and preserved in Trizol reagent. Study results revealed striking differences in the transcriptomic profiles from the three different methods that imply ex vivo changes in gene expression occurred during the blood collection, preservation, and mRNA extraction processes. When comparing the ability of the three preservation methods to accurately capture individuals' expression differences, RNAgard® outperformed PAXgene® RNA, and both showed better individual separation of transcriptomic profiles than PBMCs. Hence, our study recommends using a single blood collection platform, and strongly cautions against combining methods during the course of a defined study. [ABSTRACT FROM AUTHOR]

Malignant gliomas remain incurable with a poor prognosis despite of aggressive treatment. We have been studying the development of brain tumors in a glioma rat model, where rats develop brain tumors after prenatal exposure to ethylnitrosourea (ENU), and there is a sizable interval between when the first pathological changes are noted and tumors become detectable with MRI. Our aim to define a molecular timeline through proteomic profiling of the cerebrospinal fluid (CSF) such that brain tumor commitment can be revealed earlier than at the presymptomatic stage. A comparative proteomic approach was applied to profile CSF collected serially either before, at and after the time MRI becomes positive. Elastic net (EN) based models were developed to infer the timeline of normal or tumor development respectively, mirroring a chronology of precisely timed, "clocked", adaptations. These CSF changes were later quantified by longitudinal entropy analyses of the EN predictive metric. False discovery rates (FDR) were computed to control the expected proportion of the EN models that are due to multiple hypothesis testing. Our ENU rat brain tumor dating EN model indicated that protein content in CSF is programmed even before tumor MRI detection. The findings of the precisely timed CSF tumor microenvironment changes at presymptomatic stages, deviation from the normal development timeline, may provide the groundwork for the understanding of adaptation of the brain environment in tumorigenesis to devise effective brain tumor management strategies. [ABSTRACT FROM AUTHOR]

Network coupled cells, such as astrocytes, regulate their cellular homeostasis via Ca2+ signals spread between the cells through gap junctions. Intracellular Ca2+ release is controlled by different signaling pathways that can be stimulated by ATP, glutamate and serotonin (5-HT). Based on our findings, all these pathways are influenced by inflammatory agents and must be restored to fully recover the Ca2+ signaling network. An ultralow concentration of the local anesthetic agent bupivacaine reduced 5-HT-evoked intracellular Ca2+ release, and an ultralow concentration of the phosphodiesterase-5 inhibitor sildenafil in combination with vitamin D3 reduced ATP-evoked intracellular Ca2+ release. Combinations of these three substances downregulated 5-HT-, glutamate- and ATP-evoked intracellular Ca2+ release to a more normal Ca2+ signaling state. Furthermore, inflammatory Toll-like receptor 4 expression decreased with a combination of these three substances. Substance P receptor neurokinin (NK)-1 expression was reduced by ultralow concentrations of bupivacaine. Here, bupivacaine and sildenafil (at extremely low concentrations) combined with vitamin D3 have potential anti-inflammatory properties. According to the present study, drug combinations at the right concentrations, especially extremely low concentrations of bupivacaine and sildenafil, affect different cellular biochemical mechanisms and represent a potential solution for downregulating inflammatory parameters, thereby restoring cells or networks to normal physiological homeostasis. [ABSTRACT FROM AUTHOR]

Gastric emptying (GE) can be either delayed or accelerated in diabetes mellitus (DM). However, most research has focused on delayed GE mediated by a chronic hyperglycemic condition in DM. As such, the function of GE in the early stages of DM is not well understood. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract. In the present study, we investigated changes in GE and ICC networks in the early stages of DM using a streptozotocin-induced type 1 diabetic mouse model. The changes in GE were measured by the 13C-octanoic acid breath test. ICC networks were immunohistochemically detected by an antibody for c-Kit, a specific marker for ICC. Our results showed that GE in type 1 DM was significantly accelerated in the early stages of DM (2–4 weeks after onset). In addition, acute normalization of blood glucose levels by a single administration of insulin did not recover normal GE. ICC networks of the gastric antrum were significantly increased in DM and were not affected by the acute normalization of blood glucose. In conclusion, our results suggest that GE is accelerated in the early stages of DM, and it is associated with increased ICC networks. This mechanism may help to clarify a link between the onset of DM and GE disorders. [ABSTRACT FROM AUTHOR]

Recently, we observed a 15% decrease in the incidence of type 1 diabetes (T1D) in Australian 0-4-year-old children following the introduction of RV vaccination [[2], [3]], suggesting that RV vaccination could contribute to the primary prevention of this autoimmune disease. Australian surveillance data [[11]] show that the prevalence of RV G3 strains increased slightly along with an increase in strain diversity in the post-RV vaccine era, but G3 remains a minor component of disease-causing RV strains. RV was prevalent in nurseries, and the change to rooming-in would have altered the timing of exposure to RV, delaying it until later in the first year of life when, based on NOD mouse studies [[17]-[19]], RV might promote rather than retard development of diabetes. [Extracted from the article]

Reversal reactions (RRs) in leprosy are characterized by a reduction in the number of bacilli in lesions associated with an increase in cell-mediated immunity against the intracellular bacterium Mycobacterium leprae, the causative pathogen of leprosy. To identify the mechanisms that contribute to cell-mediated immunity in leprosy, we measured changes in the whole blood-derived transcriptome of patients with leprosy before, during and after RR. We identified an 'RR signature' of 1017 genes that were upregulated at the time of the clinical diagnosis of RR. Using weighted gene correlated network analysis (WGCNA), we detected a module of 794 genes, bisque4, that was significantly correlated with RR, of which 434 genes were part of the RR signature. An enrichment for both IFN-γ and IFN-β downstream gene pathways was present in the RR signature as well as the RR upregulated genes in the bisque4 module, including those encoding proteins of the guanylate binding protein (GBP) family that contributes to antimicrobial responses against mycobacteria. Specifically, GBP1, GBP2, GBP3 and GBP5 mRNAs were upregulated in the RR peripheral blood transcriptome, with GBP1, GBP2 and GBP5 mRNAs also upregulated in the RR disease lesion transcriptome. These data indicate that RRs involve a systemic upregulation of IFN-γ downstream genes including GBP family members as part of the host antimicrobial response against mycobacteria. [ABSTRACT FROM AUTHOR]

Background: The extent of medication adherence in patients with type 2 diabetes mellitus (T2DM) several years after starting treatment with hypoglycemic agents remains unknown. Most previous work on medication adherence targeting this group of patients has been undertaken across a single year or is questionnaire based. This study aimed to determine medication adherence status and factors affecting adherence 3 years after initiation of hypoglycemic agents, using a nationwide medical claim-based database in Japan. Methods: This retrospective study was conducted on data from 884 subjects with T2DM to better understand medication adherence, the effects of polypharmacy, and other factors. We also investigated the effects of medication nonadherence on hemoglobin A1c levels. Proportion of days covered was defined as the number of days for which a hypoglycemic agent was prescribed and in the patient’s possession to the number of days in the observation period. A proportion of days covered ≥0.8 were considered adherent, and those with a value <0.8 as nonadherence. Polypharmacy was defined as taking ≥5 medications. Results: Of the 884 patients investigated, 440 were considered adherent during the study period. Significant factors related to adherence included number of medications (3 or 4, or ≥5), male sex, age 50–<60 years, and total number of visits ≥17. Medication adherence was also a factor related to patients with hemoglobin A1c values < 7.0% at the end of the observation period. Conclusions: We surveyed medication adherence for 3 years with post medication initiation, and found that subjects aged 50–<60 years, those with ≥3 concomitant medications, and those with a total number of visits ≥17 were more likely to be adherent and persistent, and more likely to continue their hypoglycemic agents. A high degree of medication adherence was found to have a positive influence on hemoglobin A1c levels. [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]

Extrusion of xenobiotics is essential for allowing animals to remove toxic substances present in their diet or generated as a biproduct of their metabolism. By transporting a wide range of potentially noxious substrates, active transporters of the ABC transporter family play an important role in xenobiotic extrusion. One such class of transporters are the multidrug resistance P-glycoprotein transporters. Here, we investigated P-glycoprotein transport in the Malpighian tubules of the desert locust (Schistocerca gregaria), a species whose diet includes plants that contain toxic secondary metabolites. To this end, we studied transporter physiology using a modified Ramsay assay in which ex vivo Malpighian tubules are incubated in different solutions containing the P-glycoprotein substrate dye rhodamine B in combination with different concentrations of the P-glycoprotein inhibitor verapamil. To determine the quantity of the P-glycoprotein substrate extruded we developed a simple and cheap method as an alternative to liquid chromatography–mass spectrometry, radiolabelled alkaloids or confocal microscopy. Our evidence shows that: (i) the Malpighian tubules contain a P-glycoprotein; (ii) tubule surface area is positively correlated with the tubule fluid secretion rate; and (iii) as the fluid secretion rate increases so too does the net extrusion of rhodamine B. We were able to quantify precisely the relationships between the fluid secretion, surface area, and net extrusion. We interpret these results in the context of the life history and foraging ecology of desert locusts. We argue that P-glycoproteins contribute to the removal of xenobiotic substances from the haemolymph, thereby enabling gregarious desert locusts to maintain toxicity through the ingestion of toxic plants without suffering the deleterious effects themselves. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome-coronavirus (MERS-CoV) is an emerging virus that causes severe disease with fatal outcomes; however, there are currently no approved vaccines or specific treatments against MERS-CoV. Here, we developed a novel bivalent vaccine against MERS-CoV and rabies virus (RV) using the replication-incompetent P-gene-deficient RV (RVΔP), which has been previously established as a promising and safe viral vector. MERS-CoV spike glycoprotein comprises S1 and S2 subunits, with the S1 subunit being a primary target of neutralizing antibodies. Recombinant RVΔP, which expresses S1 fused with transmembrane and cytoplasmic domains together with 14 amino acids from the ectodomains of the RV-glycoprotein (RV-G), was developed using a reverse genetics method and named RVΔP-MERS/S1. Following generation of RVΔP-MERS/S1 and RVΔP, our analysis revealed that they shared similar growth properties, with the expression of S1 in RVΔP-MERS/S1-infected cells confirmed by immunofluorescence and western blot, and the immunogenicity and pathogenicity evaluated using mouse infection experiments. We observed no rabies-associated signs or symptoms in mice inoculated with RVΔP-MERS/S1. Moreover, virus-specific neutralizing antibodies against both MERS-CoV and RV were induced in mice inoculated intraperitoneally with RVΔP-MERS/S1. These findings indicate that RVΔP-MERS/S1 is a promising and safe bivalent-vaccine candidate against both MERS-CoV and RV. [ABSTRACT FROM AUTHOR]

CDGSH iron-sulfur domain-containing protein 2 (Cisd2) is pivotal to mitochondrial integrity and intracellular Ca2+ homeostasis. In the heart of Cisd2 knockout mice, Cisd2 deficiency causes intercalated disc defects and leads to degeneration of the mitochondria and sarcomeres, thereby impairing its electromechanical functioning. Furthermore, Cisd2 deficiency disrupts Ca2+ homeostasis via dysregulation of sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a) activity, resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay cardiac aging and attenuate age-related structural defects and functional decline. In addition, it results in a younger cardiac transcriptome pattern during old age. Our findings indicate that Cisd2 plays an essential role in cardiac aging and in the heart’s electromechanical functioning. They highlight Cisd2 as a novel drug target when developing therapies to delay cardiac aging and ameliorate age-related cardiac dysfunction. [ABSTRACT FROM AUTHOR]

The bipartite transcription factor β-catenin (β-cat)/T cell factor (TCF), formed by free β-cat and a given TCF family member, serves as the effector of the developmental Wnt signaling cascade. β-cat/TCFs also serve as effectors of certain peptide hormones or growth factors during adulthood. We reported that liver-specific expression of dominant-negative Transcription factor 7 like 2 (TCF7L2DN) led to impaired glucose disposal. Here we show that, in this LTCFDN transgenic mouse model, serum and hepatic lipid contents were elevated in male but not in female mice. In hepatocytes, TCF7L2DN adenovirus infection led to stimulated expression of genes that encode lipogenic transcription factors and lipogenic enzymes, while estradiol (E2) treatment attenuated the stimulation, associated with Wnt-target gene activation. Mechanistically, this E2-mediated activation can be attributed to elevated β-cat Ser675 phosphorylation and TCF expression. In wild-type female mice, ovariectomy (OVX) plus high-fat diet (HFD) challenge impaired glucose disposal and insulin tolerance, associated with increased hepatic lipogenic transcription factor sterol regulatory element-binding protein 1-c (SREBP-1c) expression. In wild-type mice with OVX, E2 reconstitution attenuated HFD-induced metabolic defects. Some of the attenuation effects, including insulin intolerance, elevated liver-weight gain, and hepatic SREBP-1c expression, were not affected by E2 reconstitution in HFD-fed LTCFDN mice with OVX. Finally, the effects of E2 in hepatocytes on β-cat/TCF activation can be attenuated by the G-protein-coupled estrogen receptor (GPER) antagonist G15. Our study thus expanded the scope of functions of the Wnt pathway effector β-cat/TCF, as it can also mediate hepatic functions of E2 during adulthood. This study also enriches our mechanistic understanding of gender differences in the risk and pathophysiology of metabolic diseases. [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]

Parkinson’s disease is associated with hyperactivity of the subthalamic nucleus (STN), contributing to motor and gait disturbances. Although deep brain stimulation of the STN alleviates certain motor dysfunction, its specific effect on gait abnormalities remains controversial. This study investigated the long-term changes in locomotion following direct infusions of botulinum toxin-A into the globus pallidus internal segment (GPi) to suppress the flow of information from the STN to the GPi in a hemiparkinsonian rat model. Static and dynamic gait parameters were quantified using a CatWalk apparatus. Interestingly, botulinum toxin-A at 0.5 ng significantly reduced only the dynamic gait parameters of hemiparkinsonian rats at 1 week and 1 month post-infusion, while static gait parameters did not change. This study offers new insights into the complexity of basal ganglia in locomotor control and shows the potential of central infusion of botulinum toxin-A as a novel intervention in the study of experimental hemiparkinson’s disease. [ABSTRACT FROM AUTHOR]

Background: The processes that result in progression of idiopathic pulmonary fibrosis (IPF) remain enigmatic. Moreover, the course of this disease can be highly variable and difficult to accurately predict. We hypothesized analyses of body mass index (BMI), a simple, routine clinical measure, may also have prognostic value in these patients, and might provide mechanistic insights. We investigated the associations of BMI changes with outcome, plasma adipokines, and adaptive immune activation among IPF patients. Methods: Data were analyzed in an IPF discovery cohort (n = 131) from the University of Pittsburgh, and findings confirmed in patients from the University of Alabama at Birmingham (n = 148). Plasma adipokines were measured by ELISA and T-cell phenotypes determined by flow cytometry. Results: Transplant-free one-year survivals in subjects with the greatest rates of BMI decrements, as percentages of initial BMI (>0.68%/month), were worse than among those with more stable BMI in both discovery (HR = 1.8, 95%CI = 1.1–3.2, p = 0.038) and replication cohorts (HR = 2.5, 95%CI = 1.2–5.2, p = 0.02), when adjusted for age, baseline BMI, and pulmonary function. BMI decrements >0.68%/month were also associated with greater mortality after later lung transplantations (HR = 4.6, 95%CI = 1.7–12.5, p = 0.003). Circulating leptin and adiponectin levels correlated with BMI, but neither adipokine was prognostic per se. BMI decrements were significantly associated with increased proportions of circulating end-differentiated (CD28null) CD4 T-cells (CD28%), a validated marker of repetitive T-cell activation and IPF prognoses. Conclusions: IPF patients with greatest BMI decrements had worse outcomes, and this effect persisted after lung transplantation. Weight loss in these patients is a harbinger of poor prognoses, and may reflect an underlying systemic process, such as adaptive immune activation. [ABSTRACT FROM AUTHOR]

Introduction: In addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. Together, platelets and neutrophils play a role in inflammation through Toll-like receptor (TLR) expression, although we do not fully understand how platelets shape neutrophil responses to TLR stimulation. Here, we aimed to determine the extent to which platelets can modulate neutrophil function in response to in vitro stimulation with TLR4, TLR2/1, and TLR2/6 agonists. Methods: Neutrophils from 10 healthy individuals were cultured alone or with autologous platelets. Neutrophils ± platelets were left unstimulated or were stimulated with 1 or 100 ng/mL lipopolysaccharide (LPS; a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist) and fibroblast-stimulating lipopeptide (FSL)-1 (a TLR2/6 agonist). Neutrophil activation and phagocytic activity were assessed by flow cytometry, and elastase and interleukin-8 secretion were assessed by ELISA. Results: The addition of platelets attenuated neutrophil CD66b and CD11b expression in response to various doses of Pam3CSK4 and FSL-1. Furthermore, platelet co-culture was associated with higher CD62L expression (indicating reduced CD62L shedding) in response to these TLR agonists. Platelets also reduced elastase secretion in unstimulated cultures and in response to low-dose TLR stimulation. Conversely, platelet co-culture increased neutrophil phagocytosis in unstimulated cultures and in response to low-dose Pam3CSK4 and FSL-1. Platelets also increased IL-8 secretion in response to low-dose LPS. Conclusion: Platelets are complex immunomodulators that can attenuate some, and simultaneously augment other, neutrophil functions. This modulation can occur both in the absence and presence of TLR stimulation. [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]

The profiles of polytraumatized patients in intensive care units were characterized. Serum and salivary markers were compared with normality between Classes I and II of APACHE II and between periods of hospitalization; these results were correlated. This was a prospective study on saliva charts and collection (n = 70). Profile: male, 27 years old, blunt traumas and collisions. Serum parameters with normality: decrease in pH, creatinine at admission to Class I, and at 48 and 72 hours in both classes; K+ at 48 h in Class II; Ca+ on admission in both classes and at 72 h in Class I. Increase in urea at 72 h in Class II, glucose at all times and in all classes, and Ca+ at 48 h in both classes. Class II had high Na+ at 48 and 72 h compared to Class I. In Class I, creatinine reduction occurred in 48 h and 72 h compared to admission and an increase of Ca+ at 48 h with admission. In Class II, pH and Na+ increased at 48 h and 72 h compared to admission. K+ decreased from admission to 48 h and increased from 48 h to 72 h. Urea increased from 48 to 72 hours. Creatinine decreased from admission to 48 and 72 hours. Ca+ increased from admission to 48 hours and decreased from 48 to 72 hours. There was an increase in the saliva levels in both classes and times in relation to normality. There was an increase in urea at admission, glucose at 72 h, and Ca+ at 48 h in Class II compared with Class I. Class I urea increased from admission to 48 h and Ca+ decreased from admission to 48 h. Class II urea decreased from 48 h to 72 h. Strong or very strong positive correlation was identified between blood and creatinine saliva at all times and regular and negative Ca+ at 72 h. This study provides evidence that salivary and serum biomarkers can be used together to monitor the evolution of the clinical symptoms of ICU patients. [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]

The general population is exposed to bisphenol A (BPA) orally, parenterally, transdermally, and environmentally as a result of the use of BPA in food packaging, plastics, and personal care products. The majority of the population nowadays (91–99%) has detectable levels of BPA inside their body. In this study, we successfully performed an inexpensive, rapid, and simple protein precipitation procedure for extraction of BPA from human plasma, followed by analysis by LC-MS/MS. This method was specifically developed for handling large numbers of samples with minimum cost and volume of sample. The developed method was accurate, precise, and reproducible for quantification of BPA from human plasma samples in the concentration range of 10–2000 ng/mL. The method was performed on samples from 150 healthy volunteers who were enrolled in the study. The mean of observed BPA level was 2.22 ± 9.91 ng/mL. Higher BPA levels were observed for females compare to that of males (p-value = 0.002), the BPA levels were higher in participants 33 years of age and older compared to those less than 33 years of age (p-value = 0.000), then the BPA levels higher in subjects with tap water as source of drinking (p-value = 0.005). This method may be valuable for general risk assessment of BPA for a large and varied population because of its efficiency and economical aspects. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify changes in skin function associated with obesity and the mechanisms underlying these changes. Functional changes and gene expression in skin were investigated in C57BL/6J mice fed either a control or high-fat diet (HFD). The insulin responsiveness of the skin and skeletal muscle was also evaluated. The effects of inhibiting insulin signaling and altered glucose concentration on skin function-associated molecules and barrier function were analyzed in keratinocytes. HFD-fed mice were not only severely obese, but also exhibited impaired skin barrier function and diminished levels of glycerol transporter aquaporin-3, keratins, and desmosomal proteins involved in maintaining skin structure. Moreover, the expression of cell cycle regulatory molecules was altered. Insulin signaling was attenuated in the skin and skeletal muscle of HFD-fed mice. In keratinocytes, inhibition of insulin signaling leads to decreased keratin expression and diminished barrier function, and higher glucose concentrations increased the expression of CDK inhibitor 1A and 1C, which are associated with cell-cycle arrest. Obesity-associated impairment of skin function can be attributed to structural fragility, abnormal glycerol transport, and dysregulated proliferation of epidermal cells. These alterations are at least partly due to cutaneous insulin resistance and hyperglycemia. [ABSTRACT FROM AUTHOR]

Trauma and hemorrhagic shock can lead to acute traumatic coagulopathy (ATC) that is not fully reversed by prehospital resuscitation as simulated with a limited volume of fresh whole blood (FWB) in a rat model. Tranexamic Acid (TXA) is used as an anti-fibrinolytic agent to reduce surgical bleeding if administered prior to or during surgery, and to improve survival in trauma if given early after trauma. It is not clear from the existing clinical literature whether TXA has the same mechanism of action in both settings. This study sought to explore the molecular mechanisms of TXA activity in trauma and determine whether administration of TXA as a supplement to FWB resuscitation could attenuate the established ATC in a rat model simulating prehospital resuscitation of polytrauma and hemorrhagic shock. In a parallel in-vitro study, the effects on clotting assays of adding plasmin at varying doses along with either simultaneous addition of TXA or pre-incubation with TXA were measured, and the results suggested that maximum anti-fibrinolytic effect of TXA on plasmin-induced fibrinolysis required pre-incubation of TXA and plasmin prior to clot initiation. In the rat model, ATC was induced by polytrauma followed by 40% hemorrhage. One hour after trauma, the rats were resuscitated with FWB collected from donor rats. Vehicle or TXA (10mg/kg) was given as bolus either before trauma (TXA-BT), or 45min after trauma prior to resuscitation (TXA-AT). The TXA-BT group was included to contrast the coagulation effects of TXA when used as it is in elective surgery vs. what is actually feasible in real trauma patients (TXA-AT group). A single dose of TXA prior to trauma significantly delayed the onset of ATC from 30min to 120min after trauma as measured by a rise in prothrombin time (PT). The plasma d-dimer as well as plasminogen/fibrinogen ratio in traumatized liver of TXA-BT were significantly lower as compared to vehicle and TXA-AT. Wet/dry weight ratio and leukocytes infiltration of lungs were significantly decreased only if TXA was administrated later, prior to resuscitation (TXA-AT). In conclusion: Limited prehospital trauma resuscitation that includes FWB and TXA may not correct established systemic ATC, but rather may improve overall outcomes of resuscitation by attenuation of acute lung injury. By contrast, TXA given prior to trauma reduced levels of fibrinolysis at the site of tissue injury and circulatory d-dimer, and delayed development of coagulopathy independent of reduction of fibrinogen levels following trauma. These findings highlight the importance of early administration of TXA in trauma, and suggest that further optimization of dosing protocols in trauma to exploit TXA’s various sites and modes of action may further improve patient outcomes. [ABSTRACT FROM AUTHOR]

Oral herpes is a highly prevalent infection caused by herpes simplex virus 1 (HSV-1). After an initial infection of the oral cavity, HSV-1 remains latent in sensory neurons of the trigeminal ganglia. Episodic reactivation of the virus leads to the formation of mucocutaneous lesions (cold sores), but asymptomatic reactivation accompanied by viral shedding is more frequent and allows virus spread to new hosts. HSV-1 DNA has been detected in many oral tissues. In particular, HSV-1 can be found in periodontal lesions and several studies associated its presence with more severe periodontitis pathologies. Since gingival fibroblasts may become exposed to salivary components in periodontitis lesions, we analyzed the effect of saliva on HSV-1 and -2 infection of these cells. We observed that human gingival fibroblasts can be infected by HSV-1. However, pre-treatment of these cells with saliva extracts from some but not all individuals led to an increased susceptibility to infection. Furthermore, the active saliva could expand HSV-1 tropism to cells that are normally resistant to infection due to the absence of HSV entry receptors. The active factor in saliva was partially purified and comprised high molecular weight complexes of glycoproteins that included secretory Immunoglobulin A. Interestingly, we observed a broad variation in the activity of saliva between donors suggesting that this activity is selectively present in the population. The active saliva factor, has not been isolated, but may lead to the identification of a relevant biomarker for susceptibility to oral herpes. The presence of a salivary factor that enhances HSV-1 infection may influence the risk of oral herpes and/or the severity of associated oral pathologies. [ABSTRACT FROM AUTHOR]

Background: Personalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection. Methods: The Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days. Results: A total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence. Conclusions: Highly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) play a critical role in host defense against influenza virus infection, and the mechanism of influenza virus to evade type I IFNs responses remains to be fully understood. Here, we found that progranulin (PGRN) was significantly increased both in vitro and in vivo during influenza virus infection. Using a PGRN knockdown assay and PGRN-deficient mice model, we demonstrated that influenza virus-inducing PGRN negatively regulated type I IFNs production by inhibiting the activation of NF-κB and IRF3 signaling. Furthermore, we showed that PGRN directly interacted with NF-κB essential modulator (NEMO) via its Grn CDE domains. We also verified that PGRN recruited A20 to deubiquitinate K63-linked polyubiquitin chains on NEMO at K264. In addition, we found that macrophage played a major source of PGRN during influenza virus infection, and PGRN neutralizing antibodies could protect against influenza virus-induced lethality in mice. Our data identify a PGRN-mediated IFN evasion pathway exploited by influenza virus with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of PGRN in innate immunity. [ABSTRACT FROM AUTHOR]

Given the association of disturbances in non-esterified fatty acid (NEFA) metabolism with the development of Type 2 Diabetes and Non-Alcoholic Fatty Liver Disease, computational models of glucose-insulin dynamics have been extended to account for the interplay with NEFA. In this study, we use arteriovenous measurement across the subcutaneous adipose tissue during a mixed meal challenge test to evaluate the performance and underlying assumptions of three existing models of adipose tissue metabolism and construct a new, refined model of adipose tissue metabolism. Our model introduces new terms, explicitly accounting for the conversion of glucose to glyceraldehye-3-phosphate, the postprandial influx of glycerol into the adipose tissue, and several physiologically relevant delays in insulin signalling in order to better describe the measured adipose tissues fluxes. We then applied our refined model to human adipose tissue flux data collected before and after a diet intervention as part of the Yoyo study, to quantify the effects of caloric restriction on postprandial adipose tissue metabolism. Significant increases were observed in the model parameters describing the rate of uptake and release of both glycerol and NEFA. Additionally, decreases in the model’s delay in insulin signalling parameters indicates there is an improvement in adipose tissue insulin sensitivity following caloric restriction. [ABSTRACT FROM AUTHOR]

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate in order to generate myofibroblasts. When MSCs were seeded in solid collagen scaffolds, they differentiated into myofibroblasts that were observed to strongly bind to the substrate, forming a 3D cell scaffold network that developed tension and shortening after KCl stimulation. Moreover, MSC-laden scaffolds recapitulated the Frank-Starling mechanism so that active tension increased in response to increases in the initial length of the contractile system. This constituted a bioengineering tissue that exhibited the contractile properties observed in both striated and smooth muscles. By using the A. F. Huxley formalism, we determined the myosin crossbridge (CB) kinetics of attachment (f1) and detachment (g1 and g2), maximum myosin ATPase activity, molar myosin concentration, unitary CB force and maximum CB efficiency. CB kinetics were dramatically slow, characterizing the non-muscle myosin type IIA (NMMIIA) present in myofibroblasts. When MSCs were seeded in solid collagen scaffolds functionalized with Arg-Gly-Asp (RGD), contractility increased and CB kinetics were modified, whereas the unitary NMMIIA-CB force and maximum CB efficiency did not change. In conclusion, we provided a non-muscle bioengineering tissue whose molecular mechanical characteristics of NMMIIA were very close to those of a non-muscle contractile tissue such as the human placenta. [ABSTRACT FROM AUTHOR]

[Fam-] trastuzumab deruxtecan (DS-8201a) is a HER2 (ERBB2)-targeting antibody-drug conjugate, composed of a HER2-targeting antibody and a topoisomerase I inhibitor, exatecan derivative, that has antitumor effects in preclinical xenograft models and clinical trials. Recently, [fam-] trastuzumab deruxtecan was reported to enhance antitumor immunity and was beneficial in combination with an anti–PD-1 antibody in a mouse model. In this study, the antitumor effect of [fam-] trastuzumab deruxtecan in combination with an anti–CTLA-4 antibody was evaluated. [Fam-] trastuzumab deruxtecan monotherapy had antitumor activity in an immunocompetent mouse model with EMT6 human HER2-expressing mouse breast cancer cells (EMT6-hHER2). [Fam-] trastuzumab deruxtecan in combination with the anti–CTLA-4 antibody induced more potent antitumor activity than that by monotherapy with either agent. The combination therapy increased tumor-infiltrating CD4+ and CD8+ T cells in vivo. Mechanistically, cured mice with treatment of [fam-] trastuzumab deruxtecan and an anti–CTLA-4 antibody completely rejected EMT6-mock cells similar to EMT6-hHER2 cells, and splenocytes from the cured mice responded to both EMT6-hHER2 and EMT6-mock cells as measured by interferon-gamma release. Taken together, these results indicate that antitumor immunity is induced by [fam-] trastuzumab deruxtecan and is facilitated in combination with anti–CTLA-4 antibody. [ABSTRACT FROM AUTHOR]

Recently, changes in urinary albumin and in GFR have been recognized as risk factors for the development of end-stage kidney disease and mortality. Though most clinical epidemiology studies of chronic kidney disease (CKD) used renal function and proteinuria at baseline alone, definitive diagnosis of CKD with multiple measurements intensifies the differences in the risk for mortality between the CKD and non-CKD populations. We hypothesized that a transient diagnosis of proteinuria and reduced renal function each indicate a significantly higher mortality compared to definitive non-CKD as the negative control and lower mortality compared with definitive CKD as the positive control. The present longitudinal study evaluated a general-population cohort of 338,094 persons who received annual health checkups, with a median 4.3-year study period. There were 2,481 deaths, including 510 CVD deaths (20.6%) and 1,328 cancer deaths (53.5%), and mortality risk was evaluated for transient proteinuria and for transiently reduced renal function. The hazard ratios (HRs) for all-cause mortality and cancer mortality were not significant, but that for cardiovascular mortality was significantly higher for transient proteinuria (HR, 1.94 [95% confidence interval, 1.27–2.96] in men and 2.78 [1.50–5.16] in women). On the other hand, transiently reduced renal function was not significant for either cardiovascular mortality risk or cancer mortality risk. We surmise that this is the first study of the mortality risk of transient dipstick proteinuria in a large general-population cohort with cause-specific death registration. Transiently positive proteinuria appears to be a significant risk specifically for cardiovascular mortality compared with definitely negative for proteinuria. [ABSTRACT FROM AUTHOR]

Objective: Although previous studies have reported a negative relationship between serum bilirubin concentration and the development of diabetes mellitus (DM), the relationship between bilirubin and insulin resistance has not been thoroughly assessed. This study was designed to determine the relationships between bilirubin, body fat distribution, and adipose tissue inflammation in patients with type 2 DM and the effect of bilirubin in an obese animal model. Method: Body fat distribution was measured using an abdominal dual bioelectrical impedance analyzer in patients with type 2 DM. We also measured glycemic control, lipid profile, serum bilirubin concentration and other clinical characteristics, and determined their relationships with body fat distribution. In the animal study, biliverdin (20 mg/kg daily) was orally administered to high-fat diet (HFD)-induced obese (DIO) mice for 2 weeks, after which intraperitoneal insulin tolerance testing was performed. Then, adipocyte area, adipocytokine expression, and macrophage polarization were evaluated in epididymal adipose tissues. Results: In the clinical study, univariate analysis showed that a lower bilirubin concentration was significantly correlated with higher body mass index, waist circumference, triglyceride, uric acid, creatinine, visceral fat area and lower HDL-C. In multivariate analyses, bilirubin concentration significantly correlated with diastolic blood pressure, creatinine, and visceral fat area. However, there was no association between bilirubin concentration and subcutaneous fat area. In the animal study, DIO mice treated with biliverdin had smaller adipocytes than untreated DIO mice and biliverdin improved HFD-induced insulin resistance. Biliverdin treatment reversed the higher gene expression of Cd11c, encoding an M1 macrophage marker, and Tnfa, encoding the proinflammatory cytokine tumor necrosis factor-α, in the adipose tissues of DIO mice. These data suggest biliverdin administration alleviates insulin resistance by ameliorating inflammation and the dysregulation of adipocytokine expression in adipose tissues of DIO mice. Conclusions: Bilirubin may protect against insulin resistance by ameliorating visceral obesity and adipose tissue inflammation. [ABSTRACT FROM AUTHOR]

Elephant camps are among the most popular destinations in Thailand for tourists from many countries. A wide range of management strategies are used by these camps, which can have varied impacts on health and welfare of elephants. The objectives of this study were to examine relationships between FGM (fecal glucocorticoid metabolite) concentrations and camp management factors (work routine, walking, restraint, rest area, foraging), and to other welfare indicators (stereotypic behaviors, body condition, foot health, and skin wounds). Data were obtained on 84 elephants (18 males and 66 females) from 15 elephant camps over a 1-year period. Elephants were examined every 3 months and assigned a body condition score, foot score, and wound score. Fecal samples were collected twice monthly for FGM analysis. Contrary to some beliefs, elephants in the observation only program where mahouts did not carry an ankus for protection had higher FGM concentrations compared to those at camps that offered riding with a saddle and shows. Elephants that were tethered in the forest at night had lower FGM concentrations compared to elephants that were kept in open areas inside the camps. There was an inverse relationship between FGM concentrations and occurrence of stereotypy, which was not anticipated. Thus, assessing adrenal activity via monitoring of FGM concentrations can provide important information on factors affecting the well-being of elephants. Results suggest that more naturalistic housing conditions and providing opportunities to exercise may be good for elephants under human care in Thailand, and that a no riding, no hook policy does not necessarily guarantee good welfare. [ABSTRACT FROM AUTHOR]

RNA contains various chemical modifications, among which N6-methyladenosine (m6A) is the most prevalent modified nucleotide in eukaryotic mRNA. Emerging evidence suggests that m6A plays an important role in regulating a variety of cellular functions by controlling mRNA processing, translation and degradation. Because m6A is not detectable by standard chemical modification-based approaches, immunological methods, such as ELISA, immunoblotting, immunohistochemistry, m6A RNA immunoprecipitation sequencing and m6A individual-nucleotide resolution cross-linking and immunoprecipitation, have been employed to detect m6A in RNA. Although the most important factor determining the success of these methods is the integrity of highly specific antibodies against m6A, the development of m6A-specific monoclonal antibodies has been challenging. We developed anti-m6A monoclonal antibodies using our recently developed single cell-based monoclonal antibody production system. The binding of one selected antibody, #B1-3, to RNA oligoribonucleotide containing a single m6A had an equilibrium dissociation constant of 6.5 nM, and this antibody exhibited negligible binding to oligoribonucleotides containing a single N1-methyladenosine and unmodified adenosine. The binding was competed by the addition of increasing concentrations of N6-methyl-ATP but not N1-methyl-ATP or ATP. Furthermore, this mAb specifically crosslinked m6A-containing oligoribonucleotide by ultraviolet light, resulting in the induction of cDNA truncation at m6A position. These results show the feasibility of using the validated m6A monoclonal antibody for the specific detection of m6A in RNA. [ABSTRACT FROM AUTHOR]

Many animals are able to sense the earth’s magnetic field, including varieties of arthropods and members of all major vertebrate groups. While the existence of this magnetic sense is widely accepted, the mechanism of action remains unknown. Building from recent work on synthetic magnetoreceptors, we propose a new model for natural magnetosensation based on the rotating magnetocaloric effect (RME), which predicts that heat generated by magnetic nanoparticles may allow animals to detect features of the earth’s magnetic field. Using this model, we identify the conditions for the RME to produce physiological signals in response to the earth’s magnetic field and suggest experiments to distinguish between candidate mechanisms of magnetoreception. [ABSTRACT FROM AUTHOR]

In this study, we analyzed PRRS virus (PRRSv) specific lymphocyte function in piglets vaccinated with Ingelvac PRRSFLEX EU® at two and three weeks of age in the presence of homologous maternal immunity. Complete analysis of maternal immunity to PRRSv was evaluated postpartum, as well as passive transfer of antibodies and T cells to the piglet through colostrum intake and before and after challenge with a heterologous PRRSv at ten weeks of age. Maternal-derived antibodies were detected in piglets but declined quickly after weaning. However, vaccinated animals restored PRRSv-specific antibody levels by anamnestic response to vaccination. Cell analysis in colostrum and milk revealed presence of PRRSv-specific immune cells at suckling with higher concentrations found in colostrum than in milk. In addition, colostrum and milk contained PRRSv-specific IgA and IgG that may contribute to protection of newborn piglets. Despite the presence of PRRSv-specific Peripheral Blood Mononuclear cells (PBMCs) in colostrum and milk, no PRRSv-specific cells could be detected from blood of the piglets at one or two weeks of life. Nevertheless, cellular immunity was detectable in pre-challenged piglets up to 7 weeks after vaccination while the non-vaccinated control group showed no interferon (IFN) γ response to PRRSv stimulation. After challenge, all piglets developed a PRRSv-specific IFNγ-response, which was more robust at significantly higher levels in vaccinated animals compared to the primary response to PRRSv in non-vaccinated animals. Cytokine analysis in the lung lumen showed a reduction of pro-inflammatory responses to PRRSv challenge in vaccinated animals, especially reduced interferon (IFN) α levels. In conclusion, vaccination of maternally positive piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX EU induced a humoral and cellular immune response to PRRSv and provided protection against virulent, heterologous PRRSv challenge. [ABSTRACT FROM AUTHOR]