Reference values of ECG parameters derived from 906 echocardiographically confirmed healthy Indian children: A population-based study from Gujarat

Introduction

There are few published studies on reference ranges of ECG parameters in children; some ethnic differences have been described.

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3–mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.Programmed necrosis or “necroptosis” has emerged in the past 5 years as a cell death mechanism that complements the conventional cell death pathway, apoptosis, in multicellular organisms. In contrast to apoptosis, necroptosis does not appear to serve an important role in multicellular organism development (1–3) but participates in the defense against pathogens and is a likely culprit in destructive inflammatory conditions (4–7). Receptor Interacting Protein Kinase-3 (RIPK3) was identified as a key effector of necroptosis in 2009 (4, 5) and its substrate, the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL), in 2012 (8, 9), but the molecular events following RIPK3-mediated phosphorylation of MLKL required to induce cell death are unclear. The RIPK1/RIPK3/MLKL necrosome has been proposed to activate PGAM5 (phosphoglycerate mutase 5) and Drp1 (Dynamin-related protein 1) to cause mitochondrial fragmentation and cell death (10), but the requirement for PGAM5, Drp1, and mitochondria for necroptosis has been questioned (1, 11–13).We described the structure of mouse MLKL revealing that MLKL contains a C-terminal pseudokinase domain and an N-terminal four-helix bundle (4HB) domain connected by a two-helix linker (the “brace” helices) (1). Based on our mutational and biochemical analyses, we proposed that the catalytically inactive pseudokinase domain functions as a molecular switch and that RIPK3-mediated phosphorylation triggers this switch by inducing a conformational change in MLKL (1, 14).Recently it has been proposed that the 4HB domain is the death effector domain within MLKL and that the killing function of MLKL relies on its oligomerization and plasma membrane association (15–18). The stoichiometry of the oligomer is, however, contentious and has been reported to contain three (15), four (16), and possibly six (17) MLKL protomers. Furthermore, several mechanisms for how this oligomer causes cell death have been proposed: Cai et al. proposed it activates the calcium channel protein Tprm7 and promotes calcium influx (15), Chen et al. showed it increased sodium influx (16), and Wang et al. proposed that the oligomerized form of MLKL has the ability to bind negatively charged lipids, including phosphoinositides and cardiolipin, which facilitates its disruption of membrane integrity (17), a model supported by a subsequent paper (18).Here, we show that the MLKL 4HB domain is sufficient to induce necroptosis and identify several charged residues clustered on two faces that are required for this function. Surprisingly the polarity of several of these charged residues is not conserved between mouse and human MLKL, and alanine substitution of negatively charged residues on the α4 helix of the 4HB domain disrupted function. This finding challenges the importance of phospholipid binding to the killing activity of the 4HB domain and illustrates that membrane association cannot solely be attributed to the interaction of poorly conserved basic residues within the MLKL 4HB domain. Intriguingly, mutation of a second cluster of residues on the 4HB domain did not preclude membrane localization or oligomerization but did prevent cell death, illustrating that additional function(s) beyond membrane translocation are required for the 4HB domain to induce cell death. MLKL oligomerization and membrane translocation were also inhibited by a small molecule, compound 1, which we identified on the basis of its affinity for the nucleotide binding site of the MLKL pseudokinase domain. These data support a model for MLKL function whereby the pseudokinase domain of MLKL holds the 4HB domain in check until phosphorylated by RIPK3, which causes a conformational change in the pseudokinase domain to unleash the 4HB domain to oligomerize and associate with membranes. Activation of MLKL can be thwarted by a small MLKL binding molecule, indicating the feasibility of targeting the nucleotide binding or “pseudoactive” sites of pseudokinases, a hitherto unexplored class of therapeutic targets.     

Sensitization,pathologic, and imaging findings comparing symptomatic and quiescent failed renal allografts

Late allograft failure (LAF) is a common cause of end stage renal disease. These patients face interrelated challenges regarding immunosuppression management, risk of graft intolerance syndrome (GIS), and sensitization. This retrospective study analyzes sensitization, pathology, imaging, and transfusion requirements in 33 LAFs presenting either with GIS (22) or grafts remaining quiescent (11). All patients underwent immunosuppression weaning to discontinuation at LAF. Profound increases in sensitization were noted for all groups and occurred in the GIS group prior to transplant nephrectomy (TxN). Patients with GIS experienced a major upswing in sensitization at, or before the time of their symptomatic presentation. For both GIS and quiescent grafts, sensitization appeared to be closely linked to immunosuppression withdrawal. Most transfusion naïve patients became highly sensitized. Fourteen patients in the GIS group underwent TxN which revealed grade II acute cellular rejection or worse, with grade 3 chronic active T‐cell‐mediated rejection. Blinded comparisons of computed tomography scan of GIS group revealed swollen allografts with fluid collections compared with the quiescent allografts (QAs), which were shrunken and atrophic. The renal volume on imaging and weight of explants nearly matched. Future studies should focus on interventions to avoid sensitization and GIS.     

Bis(Methylpyridine)‐EDTA Derivative as a Potential Ligand for PET Imaging: Synthesis,Complexation, and Biological Evaluation

A novel transitional metal ligand derivatized from EDTA‐conjugated 2‐amino‐4‐methyl pyridine, an acyclic vehicle (EDTA‐Mepy₂) was designed, synthesized, and characterized for PET imaging with ⁶⁸Ga. The drug likeliness and appropriate lipophilicity were first analyzed by molecular docking studies which shows interactive property of ligand with serum albumin protein (HSA: PDB 1E78), at Lys199, Arg257, and His242 residues, which make it more appropriate in transportation as a specific ligand for PET imaging. As a confirmation, binding constant of the ligand with human serum albumin was calculated at λ_ex = 350 nm which was found to be 4.9 × 10³ m ^?1. The pharmacokinetics of ⁶⁸Ga‐EDTA‐Mepy₂ was analyzed by blood kinetics (t_1/2 slow: 3 h 56 min and t_1/2 fast: 32 min) and biodistribution (maximum%ID/g was found in kidney at 1 h). Further the capability of this ligand was analyzed as optical marker also, by recording λ_ex = 380 nm, RFU = 8000; 710 nm, RFU = 1000 units at fixed λ_em = 280 nm. Additionally, in physiological conditions where its stability was calculated, suggests 15–20 times selectivity over the endogenously present metal ions (K_GaL/K_ZnL = 14.3, K_GaL/K_CuL = 18.1).     

Different degradation rates of nanofiber vascular grafts in small and large animal models

Nanofiber vascular grafts have been shown to create neovessels made of autologous tissue, by in vivo scaffold biodegradation over time. However, many studies on graft materials and biodegradation have been conducted in vitro or in small animal models, instead of large animal models, which demonstrate different degradation profiles. In this study, we compared the degradation profiles of nanofiber vascular grafts in a rat model and a sheep model, while controlling for the type of graft material, the duration of implantation, fabrication method, type of circulation (arterial/venous), and type of surgery (interposition graft). We found that there was significantly less remaining scaffold (i.e., faster degradation) in nanofiber vascular grafts implanted in the sheep model compared with the rat model, in both the arterial and the venous circulations, at 6 months postimplantation. In addition, there was more extracellular matrix deposition, more elastin formation, more mature collagen, and no calcification in the sheep model compared with the rat model. In conclusion, studies comparing degradation of vascular grafts in large and small animal models remain limited. For clinical translation of nanofiber vascular grafts, it is important to understand these differences.     

Recurrent preterm birth

Recurrent preterm birth is frequently defined as two or more deliveries before 37 completed weeks of gestation. The recurrence rate varies as a function of the antecedent for preterm birth: spontaneous versus indicated. Spontaneous preterm birth is the result of either preterm labor with intact membranes or preterm prelabor rupture of the membranes. This article reviews the body of literature describing the risk of recurrence of spontaneous and indicated preterm birth. Also discussed are the factors which modify the risk for recurrent spontaneous preterm birth (a short sonographic cervical length and a positive cervicovaginal fetal fibronectin test). Patients with a history of an indicated preterm birth are at risk not only for recurrence of this subtype, but also for spontaneous preterm birth. Individuals of black origin have a higher rate of recurrent preterm birth.