Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

Roscovitine targets, protein kinases and pyridoxal kinase

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.     

Mycophenolate mofetil versus azathioprine in kidney transplant recipients on steroid-free,low-dose cyclosporine immunosuppression (ATHENA): A pragmatic randomized trial

BackgroundWe compared protection of mycophenolate mofetil (MMF) and azathioprine (AZA) against acute cellular rejection (ACR) and chronic allograft nephropathy (CAN) in kidney transplant recipients on steroid-free, low-dose cyclosporine (CsA) microemulsion maintenance immunosuppression.Methods and findingsATHENA, a pragmatic, prospective, multicenter trial conducted by 6 Italian transplant centers, compared the outcomes of 233 consenting recipients of a first deceased donor kidney transplant induced with low-dose thymoglobulin and basiliximab and randomized to MMF (750 mg twice/day, n = 119) or AZA (75 to 125 mg/day, n = 114) added-on maintenance low-dose CsA microemulsion and 1-week steroid. In patients without acute clinical or subclinical rejections, CsA dose was progressively halved. Primary endpoint was biopsy-proven CAN. Analysis was by intention to treat.Participants were included between June 2007 and July 2012 and followed up to August 2016. Between-group donor and recipient characteristics, donor/recipient mismatches, and follow-up CsA blood levels were similar. During a median (interquartile range (IQR)) follow-up of 47.7 (44.2 to 48.9) months, 29 of 87 biopsied patients on MMF (33.3%) versus 31 of 88 on AZA (35.2%) developed CAN (hazard ratio (HR) [95% confidence interval (CI)]: 1.147 (0.691 to 1.904, p = 0.595). Twenty and 21 patients on MMF versus 34 and 14 on AZA had clinical [HR (95% CI): 0.58 (0.34 to 1.02); p = 0.057) or biopsy-proven subclinical [HR (95% CI): 1.49 (0.76 to 2.92); p = 0.249] ACR, respectively. Combined events [HR (95% CI): 0.85 (0.56 to 1.29); p = 0.438], patient and graft survival, delayed graft function (DGF), 3-year glomerular filtration rate (GFR) [53.8 (40.6;65.7) versus 49.8 (36.8;62.5) mL/min/1.73 m², p = 0.50], and adverse events (AEs) were not significantly different between groups.Chronicity scores other than CAN predict long-term graft outcome. Study limitations include small sample size and unblinded design.ConclusionsIn this study, we found that in deceased donor kidney transplant recipients on low-dose CsA and no steroids, MMF had no significant benefits over AZA. This finding suggests that AZA, due to its lower costs, could safely replace MMF in combination with minimized immunosuppression.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00494741","term_id":"NCT00494741"}}NCT00494741; EUDRACT 2006-005604-14.

Piero Ruggenenti and co-workers study maintenance immunosuppression in deceased-donor kidney transplantation.     

Work ability of health care shift workers: What matters?

This paper aims at identifying variables associated with inadequate work ability among nursing personnel at a public hospital, considering factors related to socio-demographic, lifestyles, working conditions, and health outcomes. A cross-sectional study was conducted in a university hospital in S?o Paulo, Brazil, as part of a larger research study on tolerance to 12 h night work. Nursing staff included registered nurses, nurse technicians, and nurse aides; in total, there were 996 healthcare workers (878 female; 118 male) at the time of the study. Some 696 workers (69.9%) of the population agreed to participate. Data collection (October 2004-July 2005) was based on a comprehensive questionnaire about living and working conditions (including incivility at work, work demands, work control, and support), mental and physical health symptoms (fatigue and sleep problems), and work ability. This report presents analyses of the adapted Brazilian version of the Work Ability Index (WAI) and associated variables. The study population worked one of the following shift schedules at this hospital: 12 h nights followed by 36 h off or 9 h or 6 h day (morning or afternoon) shifts. The mean age of the respondents was 34.9 (S.D.+/-10.4) years of age; 31.5% of the participants held two jobs. Statistical analyses using a hierarchical multiple logistic regression model were performed to evaluate the factors associated with inadequate (moderate and low scores) of the WAI. The significantly associated factors were socio-demographic (income responsibility, sole breadwinner, raising kids, age group), working conditions (thermal discomfort, organization of the workplace, and verbal abuse), and health outcomes (high body mass index, obesity, sleep problems, and fatigue). In spite of limitations of the study design, results indicate that the nursing profession is associated with stressful working conditions, contributing to inadequate WAI. This is in addition to bad living conditions and precarious work. Intervention measures, either at the workplace or at individual levels, are necessary to prevent a decrease in work ability, even in this quite young working population.     

Functionalized self-assembled monolayer on gold for detection of human mitochondrial tRNA gene mutations

We developed a rapid and simple method to identify single-nucleotide polymorphisms (SNPs) in the human mitochondrial tRNA genes. This method is based on a universal, functionalized, self-assembled monolayer, XNA on Gold chip platform. A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was immobilized on chips and the chips were then hybridized with fluorescence-labeled reference targets produced by asymmetric polymerase chain reaction from patient DNA. The ratio of the hybridization signals from the reference and test targets with each probe was then calculated. A ratio of above 3 indicates the presence of a wild-type sequence and a ratio of below 0.3 indicates a mutant sequence. We tested the sensitivity of the chip for known mutations in tRNA(Leu(UUR)) and tRNA(Lys) genes and found that it can also be used to discriminate multiple mutations and heteroplasmy, two typical features of human mitochondrial DNA. The XNA on Gold biochip method is a simple and rapid microarray method that can be used to test rapidly and reliably any SNP in the mitochondrial genome or elsewhere. It will be particularly useful for detecting SNPs associated with human diseases.     

Combined Drug Action of 2-Phenylimidazo[2,1-b]Benzothiazole Derivatives on Cancer Cells According to Their Oncogenic Molecular Signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by “RTK swapping” by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Neuronal ER Stress Impedes Myeloid-Cell-Induced Vascular Regeneration through IRE1α Degradation of Netrin-1

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Highlights? Canonical ER stress pathways are activated in central neurons during hypoxia/ischemia ? The ER stress endoribonuclease IRE1α degrades the neurovascular guidance cue netrin-1 ? Neuronal-derived netrin-1 activates a reparative proangiogenic program in microglial cells ? Neuronal ER stress prevents reparative angiogenesis in the ischemic neural retina     

A retrospective analysis of human cystic echinococcosis in Sardinia (Italy), an endemic Mediterranean region,from 2001 to 2005

To assess the current impact of human CE in Sardinia (Italy) and to monitor the changes over time, a survey has been carried out for the period 2001–2005 using hospital inpatient discharge reports (HDR) as information source, supplementing data wherever possible with additional information retrieved directly from medical records. The total of 726 admissions with “Echinococcosis” as primary diagnosis (annual rate of 8.9 per 100,000 inhabitants) concerned 540 CE cases with an annual mean incidence rate of 6.62 per 100,000 inhabitants. Male-to-female ratio was 1.36, suggesting a marked risk associated with traditional male occupations. Age-specific incidence showed increasing rates of clinical CE with age for both genders. The liver was found to be the most common localization, affecting 72% of patients, while pulmonary CE was more frequent in males than in females. CE risk was unevenly distributed in the island. The more pastoral areas had the highest probability of humans becoming infected, with an incidence rate of clinical cases of ~ 14.0 per 100,000 for areas with sheep/inhabitants index of > 6. Compared to the past, incidence rates appear to be decreasing both for pulmonary and hepatic localizations, while there is a reversal of the CE “urbanization” trend resulting in “ruralization”, accompanied by a greater degree of parasite ecological “isolation” and focus-points of infection risk. In spite of this decrease, the cost of hospital care alone (~4 million euros) suggests that the monetary plus non-monetary costs of CE are still very high but not fully recognised.