Genetic, Biochemical, and Functional Characterization of Heme Metabolism in Group A Streptococcus

Heme is vital to a variety of cellular functions in bacteria ranging from energy generation to iron reserve. Group A streptococcus (GAS) is a prevalent bacterial pathogen that is responsible for an array of human diseases ranging from simple, self-limiting, mucosal and skin infections to invasive and systemic manifestations. GAS needs iron for growth and can satisfy this nutritional requirement by scavenging the metal from heme. The pathogen produces powerful hemolysins that facilitate heme release during infection. Heme is captured and relayed through the GAS cell wall and cytoplasmic membrane by dedicated receptors and transporters. To-date, the fate of the acquired heme is unknown in Streptococci. Although heme is nutritionally beneficial for GAS growth, its pro-oxidant and lipophilic nature makes it a liability with damaging effects on cellular components. The conundrum associated with heme use is particularly pertinent to GAS pathophysiology since invasive GAS infections involve massive hemolysis and the generation of unescorted heme in excess. In this dissertation, I aimed to describe the mechanisms that GAS uses for heme catabolism while managing its toxicity. I conducted a biochemical characterization of a new enzyme, HupZ in GAS that degrades heme in vitro. Similar to the heme oxygenase-1 (HO-1), HupZ activity leads to the formation of iron, CO, and a biliverdin-like product. I also investigated the impact of heme on GAS physiology and identified key mediators in the repair and detoxification process. This study demonstrated that heme exposure leads to a general stress response that involves the activation of antioxidant defense pathways to restore redox balance. Further, I studied a 3-gene cluster, pefRCD (porphyrin-regulated efflux RCD), which was activated by environmental heme, and provided support to my hypothesis that the pefRCD gene encodes a heme-sensing regulator (PefR) and heme efflux system (PefCD). I showed that the pef system protects GAS cells from heme-induced damage to the membrane and DNA by preventing cellular accumulation of heme. In conclusion, this dissertation addresses key knowledge gaps in GAS physiology and provides new insights into heme metabolism of GAS

New distribution and host records of Chalcidoidea (Insecta: Hymenoptera) from various parts of India

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The Crimson Conundrum: Heme Toxicity and Tolerance in GAS

The massive erythrocyte lysis caused by the Group A Streptococcus (GAS) suggests that the β-hemolytic pathogen is likely to encounter free heme during the course of infection. In this study, we investigated GAS mechanisms for heme sensing and tolerance. We compared the minimal inhibitory concentration of heme among several isolates and established that excess heme is bacteriostatic and exposure to sub-lethal concentrations of heme resulted in noticeable damage to membrane lipids and proteins. Pre-exposure of the bacteria to 0.1 μM heme shortened the extended lag period that is otherwise observed when naive cells are inoculated into heme-containing medium, implying that GAS is able to adapt. The global response to heme exposure was determined using microarray analysis revealing a significant transcriptome shift that included 79 up regulated and 84 down regulated genes. Among other changes, the induction of stress-related chaperones and proteases, including groEL/ES (8x), the stress regulators spxA2 (5x) and ctsR (3x), as well as redox active enzymes were prominent. The heme stimulon also encompassed a number of regulatory proteins and two-component systems that are important for virulence. A three-gene cluster that is homologous to the pefRCD system of the Group B Streptococcus was also induced by heme. PefR, a MarR-like regulator, specifically binds heme with stoichiometry of 1:2 and protoporphyrin IX (PPIX) with stoichiometry of 1:1, implicating it is one of the GAS mediators to heme response. In summary, here we provide evidence that heme induces a broad stress response in GAS, and that its success as a pathogen relies on mechanisms for heme sensing, detoxification, and repair

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001

Effectiveness of distributed form of constraint induced movement therapy to improve functional outcome in chronic hemiparesis patients

Background:Upper limb hemiparesis is among the most common deficits after stroke that leads to disability. Learned nonuse develops due to over-reliance on the less affected limb for the functional activities. However for many stroke patients, participation in a traditional, more intense CIMT may be problematic, given the required practice intensity and the duration of the restraint schedule. So it is necessary to evaluate the effects of distributed form of Constraint Induced Movement Therapy (dCIMT) in improving functional outcome and quality of life in patients with chronic hemiparesis.Methods:36 hemiplegic patients following stroke were included. The experimental group was given dCIMT for 5sessions/week for 4 consecutive weeks in addition to conventional therapy while the control group received only conventional therapy. The outcome measures were motor activity log, wolf motor functional test and nine hole peg test.Results:The results of within group analysis for both the experimental group (Group-A) and control group B showed highly significant improvement on all the 3 outcome measures with P <0.0001.But the difference in the improvement of group-A compared to group-B was highly significant on the MAL and NHPT (P <0.0001) whereas it was not significant for WMFT performance score but highly significant for WMFT duration (U=23).Conclusion:dCIMT is an effective measure in improving the upper extremity motor function in terms of the quality and amount of use & speed and co- ordination. Thus improves the functional level and the quality of life of the patients with chronic stroke.

Oxytocin versus Misoprostol used as an induction of labour in term in early rupture of Amniotic membranes

Background: Pre labor Rupture of membranes is a common obstetrical problem, significant event as it transforms an ordinary pregnancy into a high risk one. Majority of cases of PROM - of about 60% occur after 37 completed weeks Induction of labour is artificial. Misoprostol is receiving attention as a cervical modifier and labour induction agent. This study compares the safety and efficacy of Misoprostol with Oxytocin in labour induction in term pre labour rupture of membranes. Objective of this study was to compare the safety and efficacy of Misoprostol with that of Oxytocin in labour induction in PROM. The effects were compared between primipara and multipara in a selected sample.Methods: General condition is assessed by pulse rate, blood pressure, height, weight with particular attention to pedal odema, anemia. Cardiovascular and respiratory systems were examined, rule out cephalo pelvic disproportion and for Bishop’s scoring. USG for foetal maturity, Liquor status and for foetal well-being. Admission CTG.Results: There is no significant difference was observed between two groups either in vaginal delivery or in incidence of LSCS. Mean induction delivery interval in misoprostol group for nullipara is 8.5 hours. For multipara it is 6.6 hours. And in oxytocin group for nullipara is 10:4 hours. In multipara it is 6.5 for primipara it was significantly reduced in misoprostol group compared to syntocinon group.Conclusions: Misoprostol is an effective, cheap, safe, stable at room temperature and easy to use if it is used in appropriate dosage for induction of labour in pre-labour rupture of membranes at term

Zika Virus Can Strongly Infect and Disrupt Secondary Organizers in the Ventricular Zone of the Embryonic Chicken Brain

Zika virus (ZIKV) is associated with severe neurodeve- lopmental impairments in human fetuses, including microencephaly. Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in nonuniform periventricular infec- tion 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influ- ence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8 expressed at the midbrain basal plate, hypotha- lamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced, and a SHH-dependent cell popula- tion in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non- cell-autonomous changes in brain patterning