Attenuation of cytotoxicity induced by tBHP in H9C2 cells by Bacopa monniera and Bacoside A

Cardiovascular diseases are one of the major global health issues leading to morbidity and mortality across the world. In the present study Bacopa monniera and its major bioactive component, Bacoside A (Bac-A) was used to evaluate its cytoprotective property in H9C2 cardiomyocytes against tBHP (150?μM) induced ROS-mediated oxidative stress and apoptosis. Our results implicate that pre-treatment with hydroalcoholic extract of Bacopa monniera (BME) and Bac-A (125?μg/ml and 6?μg/ml respectively) significantly restored oxidative stress by scavenging the free radicals and also elevated phase II antioxidant defensive enzymes such as (SOD, CAT, GR, GPx and GSH). Membrane integrity was estimated by MMP and LDH assays and found 89 and 72% of the protective effect. Further immunoblotting studies confirmed anti-apoptotic effects by regulating protein expression like Bcl2 was up-regulated to 99 and 85% and Bax was down-regulated to 122 and 181%, iNOS by 154.38 and 183.45% compared to tBHP (277.48%) by BME and Bac-A. BME and Bac-A exerts cytoprotective efficacy by attenuation of ROS generated through oxidative stress by an increase in the concentration of antioxidant enzymes and sustain membrane integrity which leads to restoring the damage caused by tBHP.     

Deceived,Disgusted, and Defensive: Motivated Processing of Anti-Tobacco Advertisements

A 2 × 2 experiment was conducted, where participants watched anti-tobacco messages that varied in deception (content portraying tobacco companies as dishonest) and disgust (negative graphic images) content. Psychophysiological measures, self-report, and a recognition test were used to test hypotheses generated from the motivated cognition framework. The results of this study indicate that messages containing both deception and disgust push viewers into a cascade of defensive responses reflected by increased self-reported unpleasantness, reduced resources allocated to encoding, worsened recognition memory, and dampened emotional responses compared to messages depicting one attribute or neither. Findings from this study demonstrate the value of applying a motivated cognition theoretical framework in research on responses to emotional content in health messages and support previous research on defensive processing and message design of anti-tobacco messages.     

Understanding Long-Term Outcomes Following Sepsis: Implications and Challenges

Sepsis is life-threating organ dysfunction due to infection. Incidence of sepsis is increasing and the short-term mortality is improving, generating more sepsis survivors. These sepsis survivors suffer from additional morbidities such as higher risk of readmissions, cardiovascular disease, cognitive impairment and of death, for years following index sepsis episode. In the first year following index sepsis episode, approximately 60 % of sepsis survivors have at least one rehospitalisation episode, which is most often due to infection and one in six sepsis survivors die. Sepsis survivors also have a higher risk of cognitive impairment and cardiovascular disease contributing to the reduced life expectancy seen in this population, when assessed with life table comparisons. For optimal design of interventional trials to reduce these bad outcomes in sepsis survivors, in-depth understanding of major risk factors for these morbid events, their modifiability and a causal relationship to the pathobiology of sepsis is essential. This review highlights the recent advances, clinical and methodological challenges in our understanding of these morbid events in sepsis survivors.     

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, Na_V1.5 channels mediate initiation and fast propagation of action potentials. The Ca²⁺-binding protein calmodulin (CaM) serves as a de facto subunit of Na_V1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the Na_V1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on Na_V1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca²⁺-free CaM preassociation with Na_V1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent Na_V openings. Mechanistically, these effects arise from a CaM-dependent switch in the Na_V inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant Na_V1.5, local Ca²⁺ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of Na_V1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of Na_V1.5 channelopathies.

Voltage-gated sodium channels (Na_V) are responsible for the initiation and spatial propagation of action potentials (AP) in excitable cells (1, 2). Na_V channels undergo rapid activation that underlie the AP upstroke while ensuing inactivation permits AP repolarization. The Na_V1.5 channel constitutes the predominant isoform in cardiomyocytes, whose pore-forming α-subunit is encoded by the SCN5A gene. Na_V1.5 dysfunction underlies diverse forms of cardiac disease including cardiomyopathies, arrhythmias, and sudden death (3–6). Human mutations in Na_V1.5 are associated with two forms of inherited arrhythmias–congenital long QT syndrome 3 (LQTS3) and Brugada syndrome (BrS) (7). LQTS3 stems from delayed or incomplete inactivation of Na_V1.5 that causes persistent Na influx that prolongs AP repolarization—a “gain-of-function” phenotype (7–9). BrS predisposes patients to sudden death and is associated with a reduction in the peak Na current that may slow cardiac conduction or cause region-specific repolarization differences—a “loss-of-function” phenotype (10, 11). Genetic studies have identified an expanding array of mutations in multiple Na_V1.5 domains, including the channel carboxy-terminus (CT) that is a hotspot for mutations linked to both LQTS3 and BrS (12, 13). This domain interacts with the Ca²⁺-binding protein calmodulin (CaM), suggesting that altered CaM regulation of Na_V1.5 may be a common pathophysiological mechanism (12, 14–16). More broadly, human mutations in the homologous regions of neuronal Na_V1.1 (17, 18), Na_V1.2 (19, 20), and Na_V1.6 (21) as well as skeletal muscle Na_V1.4 (22) are linked to varied clinical phenotypes including epilepsy, autism spectrum disorder, neurodevelopmental delay, and myotonia (23). Taken together, a common Na_V mechanistic deficit—defective CaM regulation—may underlie these diverse diseases.CaM regulation of Na_V channels is complex, isoform specific, and mediated by multiple interfaces within the channel (14–16). The Na_V CT consists of a dual vestigial EF hand segment and a canonical CaM-binding “IQ” (isoleucine–glutamine) domain (24, 25) (Fig. 1A). The IQ domain of nearly all Na_V channels binds to both Ca²⁺-free CaM (apoCaM) and Ca²⁺/CaM, similar to Ca_V channels (26–31). As CaM is typically a Ca²⁺-dependent regulator, much attention has been focused on elucidating Ca²⁺-dependent changes in Na_V gating. For skeletal muscle Na_V1.4, transient elevation in cytosolic Ca²⁺ causes a dynamic reduction in the peak current, a process reminiscent of Ca²⁺/CaM-dependent inactivation of Ca_V channels (32). Cardiac Na_V1.5 by comparison exhibits no dynamic effect of Ca²⁺ on the peak current (32–34). Instead, sustained Ca²⁺ elevation has been shown to elicit a depolarizing shift in Na_V1.5 steady-state inactivation (SSI or h_∞), although the magnitude and the presence of a shift have been debated (32, 35). Additional CaM-binding sites have been identified in the channel amino terminus domain (36) and the III-IV linker near the isoleucine, phenylalanine, and methionine (IFM) motif that is well recognized for its role in fast inactivation (35, 37). However, recent cryogenic electron microscopy structures, biochemical, and functional analyses suggest that both the III-IV linker and the Domain IV voltage-sensing domain might instead interact with the channel CT in a state-dependent manner (38–43).Open in a separate windowFig. 1.Absence of dynamic Ca²⁺/CaM effects on WT Na_V1.5 SSI. (A, Left) Structure of Na_V1.5 transmembrane domain (6UZ3) (70) juxtaposed with that of Na_V1.5 CT–apoCaM complex (4OVN) (28). (Right) Arrhythmia-linked CT mutations highlighted in Na_V1.5 CT–apoCaM structure (LQTS3, blue; BrS, magenta; mixed syndrome, purple). (B) Dynamic Ca²⁺-dependent changes in Na_V1.5 SSI probed using Ca²⁺ photouncaging. Na currents specifying h_∞ at ∼100 nM (Left) and ∼4 μM Ca²⁺ step (Right). (C) Population data for Na_V1.5 SSI under low (black, Left) versus high (red, Right) intracellular Ca²⁺ reveal no differences (P = 0.55, paired t test). Dots and bars are mean ± SEM (n = 8 cells). (D) FRET two-hybrid analysis of Cerulean-tagged apoCaM interaction with various Venus-tagged Na_V1.5 CT (WT, black; IQ/AA, red; S[1904]L, blue). Each dot is FRET efficiency measured from a single cell. Solid line fits show 1:1 binding isotherm.Beyond Ca²⁺-dependent effects, the loss of apoCaM binding to the Na_V1.5 IQ domain increases persistent current (34, 44), suggesting that apoCaM itself may be pathophysiologically relevant. Indeed, Na_V1.5 mutations in the apoCaM-binding interface are associated with LQTS3 and atrial fibrillation (7), as well as a loss-of-function BrS phenotype and a mixed-syndrome phenotype whereby some patients present with BrS while others with LQTS3 (Fig. 1A) (13, 45). How alterations in CaM binding paradoxically elicits both gain-of-function and loss-of-function effects is not fully understood, though important to delineate pathophysiological mechanisms and for personalized therapies.Here, using single- and multichannel recordings, we show that apoCaM binding elicits two distinct effects on Na_V1.5 gating: 1) an increase in the peak channel open probability (P_O/peak) and 2) a reduction in the normalized persistent channel open probability (R_persist), consistent with previous studies (34, 44). The two effects may explain how mixed-syndrome mutations in the Na_V1.5 CT produce either BrS or LQTS3 phenotypes. On one hand, the loss of apoCaM association may diminish P_O/peak and induce BrS by shunting cardiac AP. On the other hand, increased R_persist may prevent normal AP repolarization, resulting in LQTS3. Analysis of elementary mechanisms suggests that these changes relate to a switch in the state dependence of channel inactivation. Furthermore, dynamic changes in Ca²⁺ can inhibit persistent current for certain mutant Na_V1.5 owing to enhanced Ca²⁺/CaM binding that occurs over the timescale of a cardiac AP. This effect may result in beat-to-beat variability in persistent Na current for some mutations. In all, these findings explain how a common deficit in CaM binding can contribute to distinct arrhythmogenic mechanisms.     

Massive immature mediastinal teratoma extending into the left pleural cavity

We report a case of a massive mediastinal teratoma in an 18-year-old woman who presented with a short history of exertional dyspnoea. The tumor arose from the left lobe of the thymus and extended into the left pleural cavity, completely compressing the left lung and extensively shifting the mediastinum to the right. Measuring 23 cm x 17 cm x 9 cm and weighing 2005 g it is one of the largest anterior mediastinal teratomas reported. It was successfully treated by surgical resection, with a final pathological diagnosis of a grades 1-2 immature teratoma.     

Grading acute graft‐versus‐host disease: Time to reconsider

Acute graft‐versus‐host disease (GVHD) is a common and serious complication of allogeneic blood and marrow transplantation. Acute GVHD is commonly graded according to modified Glucksberg criteria. There is considerable within‐grade heterogeneity with different patterns of skin, liver, or gut involvement. In this commentary, we provide an analytical review of ambiguities in acute GVHD severity scoring and offer specific proposals meant to generate discussion in the BMT community for adoption, refinement, and where appropriate, validation studies.     

Early PREdiction of sepsis using leukocyte surface biomarkers: the ExPRES-sepsis cohort study

Purpose

Reliable biomarkers for predicting subsequent sepsis among patients with suspected acute infection are lacking. In patients presenting to emergency departments (EDs) with suspected acute infection, we aimed to evaluate the reliability and discriminant ability of 47 leukocyte biomarkers as predictors of sepsis (Sequential Organ Failure Assessment score?≥?2 at 24 h and/or 72 h following ED presentation).

Methods

In a multi-centre cohort study in four EDs and intensive care units (ICUs), we standardised flow-cytometric leukocyte biomarker measurement and compared patients with suspected acute infection (cohort-1) with two comparator cohorts: ICU patients with established sepsis (cohort-2), and ED patients without infection or systemic inflammation but requiring hospitalization (cohort-3).

Results

Between January 2014 and February 2016, we recruited 272, 59 and 75 patients to cohorts 1, 2, and 3, respectively. Of 47 leukocyte biomarkers, 14 were non-reliable, and 17 did not discriminate between the three cohorts. Discriminant analyses for predicting sepsis within cohort-1 were undertaken for eight neutrophil (cluster of differentiation antigens (CD) CD15; CD24; CD35; CD64; CD312; CD11b; CD274; CD279), seven monocyte (CD35; CD64; CD312; CD11b; HLA-DR; CD274; CD279) and a CD8 T-lymphocyte biomarker (CD279). Individually, only higher neutrophil CD279 [OR 1.78 (95% CI 1.23–2.57); P?=?0.002], higher monocyte CD279 [1.32 (1.03–1.70); P?=?0.03], and lower monocyte HLA-DR [0.73 (0.55–0.97); P?=?0.03] expression were associated with subsequent sepsis. With logistic regression the optimum biomarker combination was increased neutrophil CD24 and neutrophil CD279, and reduced monocyte HLA-DR expression, but no combination had clinically relevant predictive validity.

Conclusions

From a large panel of leukocyte biomarkers, immunosuppression biomarkers were associated with subsequent sepsis in ED patients with suspected acute infection.

Clinical trial registration

NCT02188992.

     

Next-Generation Sequencing Based Approach to Identify Underlying Genetic Defects of Glanzmann Thrombasthenia

Glanzmann thrombasthenia (GT) is an autosomal recessive platelet function disorder characterized by mucocutaneous bleeding as the most common clinical phenotype. Patients with GT have normal platelet counts, platelet morphology but reduced platelet aggregation in response to various agonists. Homozygosity or compound heterozygosity for variants in the ITGA2B/ITGB3 genes is the genetic basis for GT. Establishing a molecular diagnosis is definitive and is important for predictive testing. Using multi-gene panels is an accurate, faster, and cost-effective mode as compared to Sanger sequencing in large genes. We used a targeted resequencing based approach to identify pathogenic variants in eight cases in seven families. These variants were validated using Sanger sequencing in patients as well as family members and were predicted probably pathogenic using in-silico prediction tools. The variants include three missense (3/7 = 43%) (ITGA2B:c.1028 T > C, ITGA2B:c.1186G > A, ITGB3:c.1388G > C), two deletions (ITGA2B:c.559delG, ITGA2B:c.3092delT), one duplication (ITGA2B:c.1424_1427dupAGGT) and nonsense variant (ITGA2B:c.2578C > T, p.Gln860Ter). Except for one case which was compound heterozygous, the rest of the cases were homozygous. We found two novel variants that are reported for the first time in GT. The targeted resequencing based approach revealed varied genetic variants in North Indian patients, including two novels ones. The high yield of our panel indicates its suitability for usage in larger cohorts for the genetic diagnosis of GT patients. This approach is cost-effective and less cumbersome as compared to Sanger sequencing for these large size genes with multiple exons. The information so obtained is helpful in prenatal testing, carrier analysis, and genetic counseling.