A new class of bactericidal agents against S. aureus,MRSA and VRE derived from bisindolylmethane

A series of bisindolylmethanes (BIMs) (1a–7j) including hybrid BIMs 6a–6c were prepared for bioevaluation. The results of initial antimicrobial screening of compounds 1a–6c showed compounds 2b, 2m, 4a and 5b to be the most potent inhibitors, exhibiting MIC as well as MBC values equal to or less than that of ciprofloxacin (0.5–2 μg/mL) against Staphylococcus aureus, MRSA and VRE. Compound 2m was selected further to study the effect of N,N′ disubstitution towards antibacterial and antitumor activity. It was observed that substitution at N,N′ position (7a–7j) of 2m diminishes its antibacterial activity though in vitro antitumour activity against a panel of prostate, cervical and lung cancer cell lines remains more or less intact.     

Balloon mitral valvotomy: comparison between antegrade Inoue and retrograde non-transseptal techniques

AIMS: The results of percutaneous mitral valvotomy performed by theantegrade transseptal method using the Inoue balloon (n=1000;group 1) and by the retrograde non-transseptal technique usinga polyethylene balloon (n=100; group 2) were compared in a retrospective,non-randomized study. METHODS AND RESULTS: Both the groups were similar with respect to baseline characteristics.The success rate was 95% in group 1 and 93% in group 2. Therewas a significant increase in mitral valve area estimated byGorlin's equation (Group 1: from 0·8 ± 0·5to 2·1 ± 0·8 cm²; Group 2: from 0·8± 0·3 to 1·9 ± 0·8 cm², bothP<0·001) and by Doppler echocardiography using thepressure half-time method (Group 1: from 0·9 ±0·4 to 2·2 ± 0·6 cm²; Group 2: from0·9 ± 0·3 to 2·0 ± 0·7cm², both P<0·001). However, the calculated immediatepost-valvotomy mitral valve area was larger with the Inoue technique(2·1 ± 0·8 vs 1·9 ± 0·8cm²; P<0·02). Results were considered optimal whenthe mitral valve area increased to 1·5 cm², the percentageincrease was 50, and mitral regurgitation was 2/4. Out of thetotal successful procedures, optimal results were obtained in95% patients in Group 1 and 94% in Group 2. Incidence of significantmitral regurgitation (grade 3/4) was similar in two groups (Group1: 4% vs Group 2: 5%, P=ns). A significant left to right atrialshunt (Qp/Qs 1·5:1) in 2·5% and tamponade in2% of cases occurred exclusively with the Inoue technique, whileconduction disturbances, such as transient (<24 h) left bundlebranch block (28%) and complete heart block (2%) were notedwith the retrograde technique (Group 2). Local complicationswere significantly higher in Group 2 (3% vs 0·5%, P<0·01).The procedure time with the Inoue technique was shorter thanwith the retrograde (Group 1: 15 ± 8, range 10 to 35min; Group 2: 22 ± 14, range 15 to 45 min, P=0·05).Echocardiographic follow-up at 1 year showed no significantdifference in mitral valve area between the two groups (Group1 (n=300): 1·8 ± 0·8 vs Group 2 (n=60):1·9 ± 0·9 cm²; P=0·3). CONCLUSION: Balloon mitral valvotomy using the Inoue balloon and the retrogradenon-transseptal technique results in significant immediate haemodynamicand symptomatic improvement. The Inoue technique achieved alarger immediate post-valvotomy mitral valve area, but the differencewas not apparent at 1 year follow-up. Incidence of significantmitral regurgitation was similar with both the techniques; however,local complications occurred more frequently with the retrogradetechnique. Both techniques may complement each other in technicallydifficult cases.     

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.

Effects of gonadotropins and adenocorticotropin on plasmatic steroids of the catfish, Heteropneustes fossilis (Bloch).

The levels of some steroids in the plasma of female catfish were estimated following a single injection of porcine ACTH, ovine LH, or partially purified salmon gonadotropin (SG-G100) during the spawning and postspawning seasons. In the regressed catfish, injection of either LH or SG-G100 resulted in a three- to fourfold increase, compared to uninjected controls, in plasmatic cortisol for at least 1 hr. Injection of LH and ACTH resulted in two- and fourfold increases, respectively, of plasma cortisol levels as compared to saline-injected controls for both regressed and gravid fish. The concentration of plasma cortisol after ACTH treatment was higher than after LH or SG-G100. Gonadectomy did not influence the effect of LH on plasma cortisol concentration, and 20 min after injection, the cortisol concentration was identical to that of the intact fish. These results show that in the gravid catfish, as in the regressed ones, the increase in plasmatic cortisol after injection of LH or SG-G100 results principally from the activation of the interrenal. The concentrations of 11-deoxycortisol, 11-deoxycorticosterone, and 17α-hydroxy-20β-dihydroprogesterone were low in all samples and there was no evidence of an effect related to the injected hormone. Testosterone concentrations in the plasma of gravid fish injected with LH increased over the 1-hr sampling time and all values were higher than those recorded for saline- or ACTH-injected fish. Since the levels of cortisol and testosterone in the plasma of gravid catfish increase following gonadotropin administration, they may either singly or synergistically play a role in oocyte maturation.     

Use of narrow band imaging in assessing duodenal villous atrophy

Background and Objective

Narrow band imaging endoscopy with magnification (NBI-ME) has already been established in Barrett’s esophagus, stomach, and colonic mucosa, but limited work has been done in the mucosal evaluation of duodenum. A study was done to determine the correlation between NBI and histology in grading villous architecture in varied etiology.

Method

A prospective observational study comprising 105 subjects with suspected malabsorption. The presence of a diagnosed celiac disease, severe life threatening comorbidity, or pregnancy was considered as exclusion criteria. Standard endoscopy (SE), NBI-ME, multiple duodenal biopsies with histopathological examination were done in all.

Results

Fifty-one patients had celiac disease while 54 patients comprised mainly functional dyspepsia, iron deficiency anemia, tropical malabsorption syndrome, and irritable bowel syndrome. Four NBI-ME image subtypes of villous morphology have been proposed (NBI type I/II/III/IV). NBI-ME had 95 % sensitivity, 90.2 % specificity, 91.2 % positive predictive value, and 94.2 % negative predictive value for diagnosing altered villous morphology. Intraobserver kappa agreement coefficient (κ) for NBI-ME was 0.83 while interobserver agreement was 0.89 (95 % CI 0.8–0.97).

Conclusion

NBI-ME has good performance characteristics and very good kappa intra/interobserver agreement coefficient for varied subtypes of villous morphology. NBI-ME is most useful for obtaining a targeted biopsy which can be missed by conventional white light endoscopy.     

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.