Structural basis of calcineurin activation by calmodulin

Calcineurin is the only known calmodulin (CaM) activated protein phosphatase, which is involved in the regulation of numerous cellular and developmental processes and in calcium-dependent signal transduction. Although commonly assumed that CaM displaces the autoinhibitory domain (AID) blocking substrate access to its active site, the structural basis underlying activation remains elusive. We have created a fused ternary complex (CBA) by covalently linking three polypeptides: CaM, calcineurin regulatory B subunit (CnB) and calcineurin catalytic A subunit (CnA). CBA catalytic activity is comparable to that of fully activated native calcineurin in the presence of CaM. The crystal structure showed virtually no structural change in the active site and no evidence of CaM despite being covalently linked. The asymmetric unit contains four molecules; two parallel CBA pairs are packed in an antiparallel mode and the large cavities in crystal packing near the calcineurin active site would easily accommodate multiple positions of AID-bound CaM. Intriguingly, the conformation of the ordered segment of AID is not altered by CaM; thus, it is the disordered part of AID, which resumes a regular α-helical conformation upon binding to CaM, which is displaced by CaM for activation. We propose that the structural basis of calcineurin activation by CaM is through displacement of the disordered fragment of AID which otherwise impedes active site access.     

Insulin resistance and early virological response in chronic HCV infection

BackgroundStudies revealed that insulin resistance is associated with fibrosis progression and has negative impact on sustained virological response after standard antiviral therapy in patients with chronic hepatitis C (CHC).AimTo assess the role of IR on progression of liver fibrosis and early virological response (EVR) rates in patients with chronic hepatitis C infection.Patients and methodsThe study population comprised 79 subjects who underwent combination therapy for CHC. Laboratory investigations in the form of glucose, insulin, bilirubin, alanine aminotransferase (ALT), cholesterol and triglycerides and liver biopsy were done for all patients. Insulin resistance was calculated using the homeostasis model of IR (HOMA-IR).ResultsIR was increased (>2 IU) in 31 (40.7%) of patients. Early virological response was achieved among 37 patients (48.7%). No difference in EVR, viral load or grade of liver fibrosis between patients with and without IR. A significant positive correlation was found between IR and liver steatosis.ConclusionInsulin resistance is a common finding in CHC, it is associated with increase liver steatosis. However it has no impact on EVR to combined interferon ribavirin therapy, viral load or necroinflammation.     

Insulin-like Growth Factor-I Prevents the Accumulation of Autophagic Vesicles and Cell Death in Purkinje Neurons by Increasing the Rate of Autophagosome-to-lysosome Fusion and Degradation

Continuous macroautophagic activity is critical for the maintenance of neuronal homeostasis; however, unchecked or dysregulated autophagy can lead to cell death. Cultured Purkinje neurons die by an autophagy-associated cell death mechanism when deprived of trophic support. Here, we report that insulin-like growth factor-I (IGF-I) completely blocked the autophagy-associated cell death of Purkinje neurons. To examine the mechanism by which IGF-I influences autophagy, neurons were infected with adeno-RFP-LC3 and subjected to trophic factor withdrawal, and the size and number of autophagosomes were analyzed by live-cell fluorescence imaging. In control neurons, autophagy occurred at a constitutive low level with most autophagosomes measuring less than 0.75 μm. Trophic factor withdrawal increased the number and size of autophagosomes with most autophagosomes ranging between 0.75 and 1.5 μm and some reaching 1.5–2.25 μm. IGF-I added at the time of trophic factor withdrawal prevented the accumulation of the larger autophagosomes; however, it had no effect on the conversion of LC3, an indicator of autophagy induction. Instead, the rate of autophagosome-to-lysosome fusion measured by colocalization of RFP-LC3 and LysoSensor Green was accelerated by IGF-I. Treating the neurons with bafilomycin A₁ in the presence of IGF-I led to the accumulation of autophagosomes even larger than those induced by trophic factor withdrawal alone, indicating that IGF-I regulates autophagic vesicle turnover. Finally, the effect of IGF-I on autophagy was mediated by an Akt/mTOR-de pend ent and an ERK-independent pathway. These data suggest a novel role for IGF-I in protecting Purkinje neurons from autophagy-associated cell death by increasing autophagy efficiency downstream of autophagy induction.Autophagy is a regulated, catabolic pathway for the turnover of long-lived proteins, macromolecular aggregates, and damaged organelles by lysosomal degradation (1). It also plays a role in clearing the cell of invading bacteria and viruses (2, 3). In mammalian cells, the lysosomal pathway of intracellular degradation is divided into three distinct pathways: macroautophagy, microautophagy, and chaperone-mediated autophagy (4). Macroautophagy (5, 6) begins with the formation of a unique double-membrane vesicle (autophagosome) that engulfs cytoplasmic constituents such as proteins, lipids, and damaged organelles, including mitochondria. The outer membrane of the autophagosome then docks and fuses with the lysosome to deliver the sequestered cargo. The inner membrane of the fused vesicle (autolysosome) along with the interior contents of the autophagosome are degraded by lysosomal hydrolases, a process that generates nucleotides, amino acids, and free fatty acids that are recycled to provide raw materials and energy to the cell. Microautophagy (7) circumvents the autophagosome sequestration step and begins with the direct uptake of cytosolic components by invaginations and pinching off of the lysosomal membrane. As in macroautophagy, the internalized cytosolic components are digested by lysosomal enzymes, and the macromolecules are released when the vacuolar membrane disintegrates. In chaperone-mediated autophagy (8, 9) specific chaperone proteins bind to targeted proteins containing a KFERQ sequence and direct these proteins to the surface of the lysosome. These proteins bind to LAMP-2A on lysosomal membranes and are then transported across the membrane with the assistance of chaperone proteins where they are degraded by the lysosomal proteases.In eukaryotic cells, macroautophagy (hereon referred to as autophagy) occurs constitutively at low levels in most cells to perform housekeeping functions such as degradation of proteins and destruction of dysfunctional organelles. Dramatic up-regulation of autophagy occurs in the presence of external stressors such as starvation, hormonal imbalance, oxidation, extreme temperature, and infection as well as internal needs such as cellular remodeling and removal of protein aggregates (10).Dysregulated autophagy is thought to contribute to many human diseases, particularly neurodegeneration. Morphological studies of diseased human postmortem brain consistently reveal signs of enhanced autophagy in degenerating neuronal populations. Increased autophagy and disturbances in the lysosomal degradative system have been reported in many neurodegenerative conditions including Alzheimer disease (11, 12), Parkinson disease (13), Huntington disease (14), prion encephalopathies (15, 16), and diffuse Lewy body disease (17). Many of these neurodegenerative diseases are associated with the accumulation of mutant proteins. Several of the mutant proteins including α-synuclein and huntingtin are degraded by an autophagic-lysosomal pathway, suggesting that autophagy may be a protective mechanism for the clearance of toxic aggregate-prone proteins (18, 19). In mouse and Drosophila models of Huntington disease, early treatment with the mTOR inhibitor and autophagy stimulator, rapamycin, and a rapamycin analogue (CCI-779) decreased neurodegeneration and improved behavioral performance (20).In other animal models of neurodegeneration, it has been suggested that enhanced autophagy contributes to cell death. Autophagy is induced in vulnerable populations of neurons in transgenic mouse models of Alzheimer disease in which human mutant presenilin-1 and the Swedish variant of amyloid precursor protein are overexpressed (21, 22). In both conditions autophagosomes proliferate in dendrites, and the level of LC3-II, a specific marker for autophagosome formation, is elevated. This process occurs before β-amyloid pathology develops, indicating that autophagy induction is an early response in the disease. Likewise, an ultrastructural study on neuronal degeneration in transgenic mice expressing mutant human tau demonstrated the involvement of autophagic processes (23). In experimental animal models of Creutzfeldt-Jakob disease and scrapie disease, ultrastructural features of autophagy are abundant in cortical neurons (24). Extensive autophagic vacuole formation occurs in degenerating cerebellar Purkinje neurons in models of ischemia (25, 26) and in spinocerebellar ataxia 1 transgenic mice (27). Finally, neuronal death in the Lurcher mouse, which suffers from extensive Purkinje neuron degeneration caused by a point mutation in the δ2 glutamate receptor (28) appears to result from the direct activation of autophagy by Lurcher GRID2 receptors through a signaling pathway consisting of GRID-2, n-PIST, and Beclin 1 (29, 30). It is possible that autophagy is initially up-regulated in the nervous system to provide macromolecular nutrients or clear protein aggregates from neurons and that once a certain level of intracellular damage is reached, accumulation of large autophagic vacuoles exacerbate the damage eventually leading to cell death.We have established a novel model system utilizing primary cultured cerebellar neurons in which Purkinje neurons undergo up-regulation of autophagy when trophic factors are removed from their medium. The prolonged up-regulated autophagy and the accumulation of autophagic vesicles contribute to the death of these neurons, thus providing a model to examine the relationship between enhanced autophagy and cell death (31). In this report we have utilized live-cell imaging techniques to monitor autophagy in Purkinje neurons. We demonstrate that IGF-I, a potent neurotrophic factor in the cerebellum, prevents the accumulation of autophagic vesicles and cell death of Purkinje neurons by increasing the rate of autophagosome-lysosome fusion and degradation via an Akt/mTOR-dependent pathway, indicating a novel mechanism by which IGF-I maintains neuronal homeostasis.     

Effectiveness of Mechanisms and Models of Coordination between Organizations,Agencies and Bodies Providing or Financing Health Services in Humanitarian Crises: A Systematic Review

Background

Effective coordination between organizations, agencies and bodies providing or financing health services in humanitarian crises is required to ensure efficiency of services, avoid duplication, and improve equity. The objective of this review was to assess how, during and after humanitarian crises, different mechanisms and models of coordination between organizations, agencies and bodies providing or financing health services compare in terms of access to health services and health outcomes.

Methods

We registered a protocol for this review in PROSPERO International prospective register of systematic reviews under number PROSPERO2014:CRD42014009267. Eligible studies included randomized and nonrandomized designs, process evaluations and qualitative methods. We electronically searched Medline, PubMed, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL, PsycINFO, and the WHO Global Health Library and websites of relevant organizations. We followed standard systematic review methodology for the selection, data abstraction, and risk of bias assessment. We assessed the quality of evidence using the GRADE approach.

Results

Of 14,309 identified citations from databases and organizations'' websites, we identified four eligible studies. Two studies used mixed-methods, one used quantitative methods, and one used qualitative methods. The available evidence suggests that information coordination between bodies providing health services in humanitarian crises settings may be effective in improving health systems inputs. There is additional evidence suggesting that management/directive coordination such as the cluster model may improve health system inputs in addition to access to health services. None of the included studies assessed coordination through common representation and framework coordination. The evidence was judged to be of very low quality.

Conclusion

This systematic review provides evidence of possible effectiveness of information coordination and management/directive coordination between organizations, agencies and bodies providing or financing health services in humanitarian crises. Our findings can inform the research agenda and highlight the need for improving conduct and reporting of research in this field.     

Noncanonical regulation of alkylation damage resistance by the OTUD4 deubiquitinase

Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.     

High ABCC2 and Low ABCG2 Gene Expression Are Early Events in the Colorectal Adenoma-Carcinoma Sequence

Development of colorectal cancer (CRC) may result from a dysfunctional interplay between diet, gut microbes and the immune system. The ABC transport proteins ABCB1 (P-glycoprotein, Multidrug resistance protein 1, MDR1), ABCC2 (MRP2) and ABCG2 (BCRP) are involved in transport of various compounds across the epithelial barrier. Low mRNA level of ABCB1 has previously been identified as an early event in colorectal carcinogenesis (Andersen et al., PLoS One. 2013 Aug 19;8(8):e72119). ABCC2 and ABCG2 mRNA levels were assessed in intestinal tissue from 122 CRC cases, 106 adenoma cases (12 with severe dysplasia, 94 with mild-moderate dysplasia) and from 18 controls with normal endoscopy.We found significantly higher level of ABCC2 in adenomas with mild to moderate dysplasia and carcinoma tissue compared to the levels in unaffected tissue from the same individual (P = 0.037, P = 0.037, and P<0.0001) and in carcinoma and distant unaffected tissue from CRC cases compared to the level in the healthy individuals (P = 0.0046 and P = 0.036). Furthermore, ABCG2 mRNA levels were significantly lower in adenomas and carcinomas compared to the level in unaffected tissue from the same individuals and compared to tissue from healthy individuals (P<0.0001 for all). The level of ABCB2 in adjacent normal tissue was significantly higher than in tissue from healthy individuals (P = 0.011).In conclusion, this study found that ABCC2 and ABCG2 expression levels were altered already in mild/moderate dysplasia in carcinogenesis suggesting that these ABC transporters are involved in the early steps of carcinogenesis as previously reported for ABCB1. These results suggest that dysfunctional transport across the epithelial barrier may contribute to colorectal carcinogenesis.     

Human monocytes augment invasiveness and proteolytic activity of inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer, and here, we examined in vitro the interactions between the human IBC cell line SUM149 and U937 human naive monocytes. We found an altered morphology, enhanced invasiveness and proteolytic activity of SUM149 cells when cultured with U937 cells or in U937-conditioned media (U937-CM). Increases in expression and activity of the cysteine protease cathepsin B and expression of caveolin-1 were also detected in SUM149 cells grown in U937-CM, thus suggesting a contribution of these proteins to the augmented invasion through and proteolysis of the extracellular matrix by the IBC cells.     

The expression of interleukin-8 and interleukin-8 receptors in endometrial carcinoma

Interleukin-8 (IL-8) is a pro-inflammatory cytokine which exerts its effects via binding to 2 receptors, CXCR1 and CXCR2 and is known to promote angiogenesis, mitogenesis and motogenesis in cancer. IL-8 is over expressed in endometrial carcinoma, but the expression of CXCR1 and CXCR2 in endometrial carcinoma has not been previously investigated. The aim of this study was to determine the expression of IL-8 receptors in endometrial carcinoma. The expression of CXCR1 and CXCR2 was studied in endometrial carcinomas and normal endometrium by immunohistochemistry in 101 tumours. IL-8 and IL-8 receptor expression was also studied by Real-time quantitative PCR (RT-qPCR) in 17 tumours in comparison to normal endometrium. The expression profile was correlated to the clinico-pathological features of the tumours. Immunohistochemistry showed CXCR1 and CXCR2 were expressed in all cases of endometrial carcinoma, with CXCR1 showing stronger expression. There was a statistically significant correlation between CXCR2 staining intensity and tumour grade (P=0.012) and disease free survival (P=0.015) independently. On RT-qPCR, 14/17, 15/17 and 16/17 tumours showed significant increase in IL-8, CXCR1 and CXCR2 expression levels in comparison to normal endometrium, with median fold increase of 42-fold, 51-fold and 27-fold, respectively. This is the first report of the expression of IL-8 receptors in endometrial carcinoma and the results show an association between IL-8 and IL-8 receptors and the pathogenesis of endometrial carcinoma, and represent potential prognostic biomarkers and therapeutic targets.     

Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.