Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death

Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster. However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo. Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3, but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.     

Perceived Morbidity,Healthcare-Seeking Behavior and Their Determinants in a Poor-Resource Setting: Observation from India

BackgroundTo control the double burden of communicable and non-communicable diseases (NCDs), in the developing world, understanding the patterns of morbidity and healthcare-seeking is critical. The objective of this cross-sectional study was to determine the distribution, predictors and inter-relationship of perceived morbidity and related healthcare-seeking behavior in a poor-resource setting.MethodsBetween October 2013 and July 2014, 43999 consenting subjects were recruited from 10107 households in Malda district of West Bengal state in India, through multistage random sampling, using probability proportional-to-size. Information on socio-demographics, behaviors, recent ailments, perceived severity and healthcare-seeking were analyzed in SAS-9.3.2.ResultsRecent illnesses were reported by 55.91% (n=24600) participants. Among diagnosed ailments (n=23626), 50.92% (n=12031) were NCDs. Respiratory (17.28%,n=7605)), gastrointestinal (13.48%,n=5929) and musculoskeletal (6.25%,n=2749) problems were predominant. Non-qualified practitioners treated 53.16% (n=13074) episodes. Older children/adolescents [adjusted odds ratio for private healthcare providers(AOR_Pri)=0.76, 95% confidence interval=0.71-0.83) and for Govt. healthcare provider(AOR_Govt)=0.80(0.68-0.95)], females [AOR_Govt=0.80(0.73-0.88)], Muslims [AOR_Pri=0.85(0.69-0.76) and AOR_Govt=0.92(0.87-0.96)], backward castes [AOR_Govt=0.93(0.91-0.96)] and rural residents [AOR_Pri=0.82(0.75-0.89) and AOR_Govt=0.72(0.64-0.81)] had lower odds of visiting qualified practitioners. Apparently less severe NCDs [acid-peptic disorders: AOR_Pri=0.41(0.37-0.46) & AOR_Govt=0.41(0.37-0.46), osteoarthritis: AOR_Pri=0.72(0.59-0.68) & AOR_Govt=0.58(0.43-0.78)], gastrointestinal [AOR_Pri=0.28(0.24-0.33) & AOR_Govt=0.69(0.58-0.81)], respiratory [AOR_Pri=0.35(0.32-0.39) & AOR_Govt=0.46(0.41-0.52)] and skin infections [AOR_Pri=0.65(0.55-0.77)] were also less often treated by qualified practitioners. Better education [AOR_Pri=1.91(1.65-2.22) for ≥graduation], sanitation [AOR_Pri=1.58(1.42-1.75)] and access to safe water [AOR_Pri=1.33(1.05-1.67)] were associated with healthcare-seeking from qualified private practitioners. Longstanding NCDs [chronic obstructive pulmonary diseases: AOR_Pri=1.80(1.46-2.23), hypertension: AOR_Pri=1.94(1.60-2.36), diabetes: AOR_Pri=4.94(3.55-6.87)] and serious infections [typhoid: AOR_Pri=2.86(2.04-4.03)] were also more commonly treated by qualified private practitioners. Potential limitations included temporal ambiguity, reverse causation, generalizability issues and misclassification.ConclusionIn this poor-resource setting with high morbidity, ailments and their perceived severity were important predictors for healthcare-seeking. Interventions to improve awareness and healthcare-seeking among under-privileged and vulnerable population with efforts to improve the knowledge and practice of non-qualified practitioners probably required urgently.     

Developmental changes in the chromatin of the brain of the rat: analysis by nicktranslation

Nick translation of nuclei of the brain of 3- 14- and 30-day old rats was carried out following their digestion by DNase I. The incorporation of ³H-dTMP at 14- and 30-day is significantly lower than at 3-day. This may be due to a lower proportion of active chromatin (DNase I hypersensitive sites) and condensation of chromatin with progressive development. When nuclei were digested by EcoRI and then nick-translated, the incorporation of ³H-dTMP showed the same pattern. Since the EcoRI sites are believed to be randomly distributed, the overall conformation of chromatin including the DNase I sensitive sites seems to undergo increasing compaction with development.     

Inhibition of apoptosome activation protects injured motor neurons from cell death

Within the mammalian central nervous system many forms of neurodegenerative injury are regulated via programmed cell death, a highly conserved program of cellular suicide. Programmed cell death is regulated by multiple signaling pathways, which have been identified within mammalian cells, although several lines of evidence suggest that the intrinsic pathway predominantly regulates the death of motor neurons following acute injury in vivo. We have tested this hypothesis by performing facial axotomies on cytochrome c knock-in mice containing a point mutation in the genomic locus of cytochrome c resulting in a lysine to alanine conversion at position 72 of the protein. The introduced mutation inhibits the ability of cytochrome c to induce the formation of the apoptosome, a protein complex that is principally required for the activation of the intrinsic pathway, but does not alter its function in oxidative phosphorylation. Homozygous cytochrome c knock-in mutants displayed a significant enhancement in motor neuron survival following injury when compared with littermate controls, thus establishing the apoptosome as a viable target for protecting motor neurons from neural injury. However, protection of facial motor neurons differs from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family, which are thought to regulate several aspects of mitochondrial dysfunction including the release of cytochrome c from the mitochondria to the cytoplasm. Therefore, these results directly demonstrate for the first time the influence of the apoptosome on injury-induced neuronal programmed cell death in vivo isolated from upstream Bcl-2 family-mediated effects.     

A Rare Case Report of Subcutaneous Phaeohyphomycotic Cyst Caused by Exophiala oligosperma in an Immunocompetent Host with Literature Review

We report a rare case of phaeohyphomycotic cyst in an immunocompetent patient caused by Exophiala oligosperma. This fungus is earlier known to cause infections in the immunocompromised. Identification of black fungi at species level is more challenging by conventional methods, and hence final identification of the fungi was based on sequencing of rDNA. The patient was managed with surgical excision. To the best of our knowledge, this is the first case report of E. oligosperma human infection from India.     

Zebrafish Rohon-Beard Neuron Development: Cdk5 in the Midst

Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase that is activated mostly by association with its activators, p35 and p39. Initially projected as a neuron-specific kinase, cdk5 is expressed ubiquitously and its kinase activity solely depends on the presence of its activators, which are also found in some non-neuronal tissues. As a multifunctional protein, cdk5 has been linked to axonogenesis, cell migration, exocytosis, neuronal differentiation and apoptosis. Cdk5 plays a critical role in functions other than normal physiology, especially in neurodegeneration. Its contribution to both normal physiological as well as pathological processes is mediated by its specific substrates. Cdk5-null mice are embryonically lethal, therefore making it difficult to study precisely what cdk5 does to the nervous system at early stages of development, be it neuron development or programmed cell death. Zebrafish model system bypasses the impediment, as it is amenable to reverse genetics studies. One of the functions that we have followed for the cdk5 ortholog in zebrafish in vivo is its effect on the Rohon-Beard (RB) neurons. RB neurons are the primary sensory spinal neurons that die during the first two days of zebrafish development eventually to be replaced by the dorsal root ganglia (DRG). Based on ours studies and others’, here we discuss possible mechanisms that may be involved in cdk5’s role in RB neuron development and survival.