Deciphering a mechanism of membrane permeabilization by α-hordothionin peptide

α-Hordothionin (αHTH) belongs to thionins, the plant antimicrobial peptides with membrane-permeabilizing activity which is associated with broad-range antimicrobial activity. Experimental data have revealed a phospholipid-binding site and indicated formation of ion channels as well as membrane disruption activity of thionin. However, the mechanism of membrane permeabilization by thionin remained unknown. Here it is shown that thionin is a small water-selective channel. Unbiased high-precision molecular modeling revealed formation of a water-selective pore running through the αHTH double α-helix core when the peptide interacted with anions. Anion-induced unfolding of the C-end of the α2-helix opened a pore mouth. The pore started at the α2 C-end between the hydrophilic and the hydrophobic regions of the peptide surface and ended in the middle of the unique hydrophobic region at the C-end of the α1-helix. Highly conserved residues including cysteines and tyrosine lined the pore walls. A large positive electrostatic potential accumulated inside the pore. The narrow pore was, nonetheless, sufficient to accommodate at least one water molecule along the channel except for two constriction sites. Both constriction sites were formed by residues participating in the phospholipid-binding site. The channel properties resembled that of aquaporins with two selectivity filters, one at the entrance, inside the α2 C-end cavity, and a second in the middle of the channel. It is proposed that the αHTH water channel delivers water molecules to the bilayer center that leads to local membrane disruption. The proposed mechanism of membrane permeabilization by thionins explains seemingly controversial experimental data.     

Survival of civilian and prisoner drug-sensitive, multi- and extensive drug- resistant tuberculosis cohorts prospectively followed in Russia

Objective and Methods

A long-term observational study was conducted in Samara, Russia to assess the survival and risk factors for death of a cohort of non-multidrug resistant tuberculosis (non-MDRTB) and multidrug resistant tuberculosis (MDRTB) civilian and prison patients and a civilian extensive drug-resistant tuberculosis (XDRTB) cohort.

Results

MDRTB and XDRTB rates of 54.8% and 11.1% were identified in the region. Half (50%) of MDRTB patients and the majority of non-MDRTB patients (71%) were still alive at 5 years. Over half (58%) of the patients died within two years of establishing a diagnosis of XDRTB. In the multivariate analysis, retreatment (HR = 1.61, 95%CI 1.04, 2.49) and MDRTB (HR = 1.67, 95%CI 1.17, 2.39) were significantly associated with death within the non-MDR/MDRTB cohort. The effect of age on survival was relatively small (HR = 1.01, 95%CI 1.00, 1.02). No specific factor affected survival of XDRTB patients although median survival time for HIV-infected versus HIV-negative patients from this group was shorter (185 versus 496 days). The majority of MDRTB and XDRTB strains (84% and 92% respectively) strains belonged to the Beijing family. Mutations in the rpoB (codon 531 in 81/92; 88.8%), katG (mutation S315T in 91/92, 98.9%) and inhA genes accounted for most rifampin and isoniazid resistance respectively, mutations in the QRDR region of gyrA for most fluroquinolone resistance (68/92; 73.5%).

Conclusions

Alarmingly high rates of XDRTB exist. Previous TB treatment cycles and MDR were significant risk factors for mortality. XDRTB patients'' survival is short especially for HIV-infected patients. Beijing family strains comprise the majority of drug-resistant strains.     

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H₂O₂ in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H₂O₂ levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H₂O₂ is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).     

Synthesis and biological evaluation of novel mono- and bivalent ASGP-R-targeted drug-conjugates

Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug – paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.     

Laboratory Test Surveillance following Acute Kidney Injury

Background

Patients with hospitalized acute kidney injury (AKI) are at increased risk for accelerated loss of kidney function, morbidity, and mortality. We sought to inform efforts at improving post-AKI outcomes by describing the receipt of renal-specific laboratory test surveillance among a large high-risk cohort.

Methods

We acquired clinical data from the Electronic health record (EHR) of 5 Veterans Affairs (VA) hospitals to identify patients hospitalized with AKI from January 1^st, 2002 to December 31^st, 2009, and followed these patients for 1 year or until death, enrollment in palliative care, or improvement in renal function to estimated GFR (eGFR) ≥60 L/min/1.73 m². Using demographic data, administrative codes, and laboratory test data, we evaluated the receipt and timing of outpatient testing for serum concentrations of creatinine and any as well as quantitative proteinuria recommended for CKD risk stratification. Additionally, we reported the rate of phosphorus and parathyroid hormone (PTH) monitoring recommended for chronic kidney disease (CKD) patients.

Results

A total of 10,955 patients admitted with AKI were discharged with an eGFR<60 mL/min/1.73 m². During outpatient follow-up at 90 and 365 days, respectively, creatinine was measured on 69% and 85% of patients, quantitative proteinuria was measured on 6% and 12% of patients, PTH or phosphorus was measured on 10% and 15% of patients.

Conclusions

Measurement of creatinine was common among all patients following AKI. However, patients with AKI were infrequently monitored with assessments of quantitative proteinuria or mineral metabolism disorder, even for patients with baseline kidney disease.     

Genetic dissection of collagen-induced arthritis in Chromosome 10 quantitative trait locus speed congenic rats: evidence for more than one regulatory locus and sex influences

Rat Chromosome 10 (RNO10) harbors Cia5, a non-MHC quantitative trait locus (QTL) that regulates the severity of type II collagen-induced arthritis (CIA) in DAxF344 and DAxBN F2 rats. CIA is an animal model with many features that resemble rheumatoid arthritis. To facilitate analysis of Cia5 independently of the other CIA regulatory loci on other chromosomes, DA recombinant QTL speed congenic rats, DA.F344(Cia5), were generated. These QTL congenic rats have a large chromosomal segment containing Cia5 (interval size < or =80.1 cM) from CIA-resistant F344 rats introgressed into their genome. Phenotypic analyses of these rats for susceptibility and severity of CIA confirmed that Cia5 is an important disease-modifying locus. CIA severity was significantly lower in the Cia5 congenic rats than in DA controls. We also generated DA Cia5 speed sub-congenic rats, DA.F344(Cia5a), which had a smaller segment of the F344 genome, Cia5a, comprising only the distal q-telomeric end (interval size < or = 22.5 cM) of Cia5, introgressed into their genome. DA.F344(Cia5a) sub-congenic rats also exhibited reduced CIA disease severity compared with the parental DA rats. The regulatory effects in both congenic strains were sex influenced. The disease-ameliorating effect of the larger fragment, Cia5, was greater in males than in females, but the effect of the smaller fragment, Cia5a, was greater in females. We also present an improved genetic linkage map covering the Cia5/Cia5a region, which we have integrated with two rat radiation hybrid maps. Comparative homology analysis of this genomic region with mouse and human chromosomes was also undertaken. Regulatory loci for multiple autoimmune/inflammatory diseases in rats (RNO10), mice (MMU11), and humans (HSA17 and HSA5q23-q31) map to chromosomal segments homologous to Cia5 and Cia5a.     

Diversity,migration routes,and worldwide population genetic structure of Lecanosticta acicola,the causal agent of brown spot needle blight

Lecanosticta acicola is a pine needle pathogen causing brown spot needle blight that results in premature needle shedding with considerable damage described in North America, Europe, and Asia. Microsatellite and mating type markers were used to study the population genetics, migration history, and reproduction mode of the pathogen, based on a collection of 650 isolates from 27 countries and 26 hosts across the range of L. acicola. The presence of L. acicola in Georgia was confirmed in this study. Migration analyses indicate there have been several introduction events from North America into Europe. However, some of the source populations still appear to remain unknown. The populations in Croatia and western Asia appear to originate from genetically similar populations in North America. Intercontinental movement of the pathogen was reflected in an identical haplotype occurring on two continents, in North America (Canada) and Europe (Germany). Several shared haplotypes between European populations further suggests more local pathogen movement between countries. Moreover, migration analyses indicate that the populations in northern Europe originate from more established populations in central Europe. Overall, the highest genetic diversity was observed in south‐eastern USA. In Europe, the highest diversity was observed in France, where the presence of both known pathogen lineages was recorded. Less than half of the observed populations contained mating types in equal proportions. Although there is evidence of some sexual reproduction taking place, the pathogen spreads predominantly asexually and through anthropogenic activity.     

Fluorescence in situ hybridization test for detection of endometrial carcinoma cells by non-invasive vaginal swab

Endometrial cancer (EC) is the most common gynaecological malignancy with increasing incidence in developed countries. As gold standard, hysteroscopy confirms only 30% of suspected ECs. The detection of EC cells in the vagina by fluorescence in situ hybridization (FISH) after a smear test could reduce invasive procedures in the future. Using array-based comparative genome hybridization (aCGH) on 65 endometrial carcinomas, most frequently imbalanced regions of the tumour genome were identified. Bacterial artificial chromosomes were used to generate FISH-probes homologue to these human regions. The FISH test was hybridized on swabs specimens collected from the vaginal cavity. Samples from six patients without EC were selected as a negative control and on 13 patients with known EC as a positive control. To distinguish between benign and EC cases, the cut-off value has been defined. A first validation of this EC-FISH Test was performed with swabs from 41 patients with suspected EC. The most common genomic imbalances in EC are around the CTNNB1, FBXW7 and APC genes. The cut-off is defined at 32% of analysed cells without diploid signal pattern. This differs significantly between the positive and negative controls (p < 0.001). In a first validation cohort of 41 patients with suspected EC, the EC-FISH Test distinguishes patients with and without EC with a sensitivity of 91% and a specificity of 83%. The negative predictive value is 96%. This is the first report of a non-invasive EC-FISH Test to predict EC in women with suspected EC.     

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.