Structural Analysis and Diversity of Calmodulin-Binding Domains in Membrane and Intracellular Ca2+-ATPases.

The plasma membrane and autoinhibited Ca2+-ATPases contribute to the Ca2+ homeostasis in a wide variety of organisms. The enzymatic activity of these pumps is stimulated by calmodulin, which interacts with the target protein through the calmodulin-binding domain (CaMBD). Most information about this region is related to all calmodulin modulated proteins, which indicates general chemical properties and there is no established relation between Ca2+ pump sequences and taxonomic classification. Thus, the aim of this study was to perform an in silico analysis of the CaMBD from several Ca2+-ATPases, in order to determine their diversity and to detect specific patterns and amino acid selection in different species. Patterns related to potential and confirmed CaMBD were detected using sequences retrieved from the literature. The occurrence of these patterns was determined across 120 sequences from 17 taxonomical classes, which were analyzed by a phylogenetic tree to establish phylogenetic groups. Predicted physicochemical characteristics including hydropathy and net charge were calculated for each group of sequences. 22 Ca2+-ATPases sequences from animals, unicellular eukaryotes, and plants were retrieved from bioinformatic databases. These sequences allow us to establish the Patterns 1(GQILWVRGLTRLQTQ), 3(KNPSLEALQRW), and 4(SRWRRLQAEHVKK), which are present at the beginning of putative CaMBD of metazoan, parasites, and land plants. A pattern 2 (IRVVNAFR) was consistently found at the end of most analyzed sequences. The amino acid preference in the CaMBDs changed depending on the phylogenetic groups, with predominance of several aliphatic and charged residues, to confer amphiphilic properties. The results here displayed show a conserved mechanism to contribute to the Ca2+ homeostasis across evolution and may help to detect putative CaMBDs.

Benchtop nuclear magnetic resonance-based metabolomic approach for the diagnosis of bovine tuberculosis.

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non-tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high-field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low-field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB-vaccinated healthy control (n = 10) and healthy PTB-unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77-1). In summary, plasma fingerprinting using HF and LF-NMR differentiated TB subjects from uninfected animals, and PTB and PTB-vaccinated subjects who may provide a TB-false positive, highlighting the use of LF-NMR-based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods.

A store-operated Ca2+-entry in Trypanosoma equiperdum: Physiological evidences of its presence.

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca2+ entry are scarce. Even more, to date the presence of a store-operated Ca2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca2+-entry (SOCE) in T. equiperdum, using physiological techniques. This Ca2+-entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum.

Serological technique for detecting tuberculosis prevalence in sheep in Atlantic Spain.

Recent studies show that sheep could be considered to be a maintenance host for the causative agents of animal tuberculosis (TB). The performance of diagnostic tests is not well established, and new tests need to be developed for this species. In addition, information about TB prevalence in sheep is scarce. Our objectives were to evaluate a new P22 ELISA for detection of specific antibodies against Mycobacterium tuberculosis Complex (MTC), and to assess the seropositivity in 3998 sheep from herds sampled in TB hotspot areas of northern Atlantic Spain with a low TB prevalence in cattle. Results based on 80 sheep of known infection status suggest excellent sensitivity and specificity (100% and 98%, respectively) even in a M. avium susbsp. paratuberculosis infected flock. The observed TB seroprevalence was 17.96% (698/3998; CI95% 16.31-18.67). Our results indicate that the P22 ELISA may constitute a good option for TB screening at the herd level in sheep, and that sheep are an important host and control programs should be implemented at least in hotspots or when cohabiting with other TB-infected species, i.e. cattle and goats.

Remote Treatment Monitoring on Hospitalization and Technique Failure Rates in Peritoneal Dialysis Patients.

Background: An integrated kidney disease healthcare company implemented a peritoneal dialysis (PD) remote treatment monitoring (RTM) application in 2016. We assessed if RTM utilization associates with hospitalization and technique failure rates. Methods: We used data from adult (age ≥18 years) patients on PD treated from October 2016 through May 2019 who registered online for the RTM. Patients were classified by RTM use during a 30-day baseline after registration. Groups were: nonusers (never entered data), moderate users (entered one to 15 treatments), and frequent users (entered >15 treatments). We compared hospital admission/day and sustained technique failure (required >6 consecutive weeks of hemodialysis) rates over 3, 6, 9, and 12 months of follow-up using Poisson and Cox models adjusted for patient/clinical characteristics. Results: Among 6343 patients, 65% were nonusers, 11% were moderate users, and 25% were frequent users. Incidence rate of hospital admission was 22% (incidence rate ratio [IRR]=0.78; P=0.002), 24% (IRR=0.76; P<0.001), 23% (IRR=0.77; P≤0.001), and 26% (IRR=0.74; P≤0.001) lower in frequent users after 3, 6, 9, and 12 months, respectively, versus nonusers. Incidence rate of hospital days was 38% (IRR=0.62; P=0.013), 35% (IRR=0.65; P=0.001), 34% (IRR=0.66; P≤0.001), and 32% (IRR=0.68; P<0.001) lower in frequent users after 3, 6, 9, and 12 months, respectively, versus nonusers. Sustained technique failure risk at 3, 6, 9, and 12 months was 33% (hazard ratio [HR]=0.67; P=0.020), 31% (HR=0.69; P=0.003), 31% (HR=0.69; P=0.001), and 27% (HR=0.73; P=0.001) lower, respectively, in frequent users versus nonusers. Among a subgroup of survivors of the 12-month follow-up, sustained technique failure risk was 26% (HR=0.74; P=0.023) and 21% (HR=0.79; P=0.054) lower after 9 and 12 months, respectively, in frequent users versus nonusers. Conclusions: Our findings suggest frequent use of an RTM application associates with less hospital admissions, shorter hospital length of stay, and lower technique failure rates. Adoption of RTM applications may have the potential to improve timely identification/intervention of complications.

Seasonal and Secular Trends of Cardiovascular, Nutritional, and Inflammatory Markers in Patients on Hemodialysis.

Background: All life on earth has adapted to the effects of changing seasons. The general and ESKD populations exhibit seasonal rhythms in physiology and outcomes. The ESKD population also shows secular trends over calendar time that can convolute the influences of seasonal variations. We conducted an analysis that simultaneously considered both seasonality and calendar time to isolate these trends for cardiovascular, nutrition, and inflammation markers. Methods: We used data from adult patients on hemodialysis (HD) in the United States from 2010 through 2014. An additive model accounted for variations over both calendar time and time on dialysis. Calendar time trends were decomposed into seasonal and secular trends. Bootstrap procedures and likelihood ratio methods tested if seasonal and secular variations exist. Results: We analyzed data from 354,176 patients on HD at 2436 clinics. Patients were 59±15 years old, 57% were men, and 61% had diabetes. Isolated average secular trends showed decreases in pre-HD systolic BP (pre-SBP) of 2.6 mm Hg (95% CI, 2.4 to 2.8) and interdialytic weight gain (IDWG) of 0.35 kg (95% CI, 0.33 to 0.36) yet increases in post-HD weight of 2.76 kg (95% CI, 2.58 to 2.97). We found independent seasonal variations of 3.3 mm Hg (95% CI, 3.1 to 3.5) for pre-SBP, 0.19 kg (95% CI, 0.17 to 0.20) for IDWG, and 0.62 kg (95% CI, 0.46 to 0.79) for post-HD weight as well as 0.12 L (95% CI, 0.11 to 0.14) for ultrafiltration volume, 0.41 ml/kg per hour (95% CI, 0.37 to 0.45) for ultrafiltration rates, and 3.30 (95% CI, 2.90 to 3.77) hospital days per patient year, which were higher in winter versus summer. Conclusions: Patients on HD show marked seasonal variability of key indicators. Secular trends indicate decreasing BP and IDWG and increasing post-HD weight. These methods will be of importance for independently determining seasonal and secular trends in future assessments of population health.

Longitudinal patterns of health-related quality of life and dialysis modality: a national cohort study.

BACKGROUND: Health-related quality of life (HrQoL) varies among dialysis patients. However, little is known about the association of dialysis modality with HrQoL over time. We describe longitudinal patterns of HrQoL among chronic dialysis patients by treatment modality. METHODS: National retrospective cohort study of adult patients who initiated in-center dialysis or a home modality (peritoneal or home hemodialysis) between 1/2013 and 6/2015. Patients remained on the same modality for the first 120 days of the first two years. HrQoL was assessed by the Kidney Disease and Quality of Life-36 (KDQOL) survey in the first 120 days of the first two years after dialysis initiation. Home modality patients were matched to in-center patients in a 1:5 fashion. RESULTS: In-center (n=4234) and home modality (n=880) patients had similar demographic and clinical characteristics. In-center dialysis patients had lower mean KDQOL scores across several domains compared to home modality patients. For patients who remained on the same modality, there was no change in HrQoL. However, there were trends towards clinically meaningful changes in several aspects of HrQoL for patients who switched modalities. Specifically, physical functioning decreased for patients who switched from home to in-center dialysis (p< 0.05). CONCLUSIONS: Among a national cohort of chronic dialysis patients, there was a trend towards different patterns of HrQoL life that were only observed among patients who changed modality. Patients who switched from home to in-center modalities had significant lower physical functioning over time. Providers and patients should be mindful of HrQoL changes that may occur with dialysis modality change.

Tract dilation to salvage failing buttonholes in arteriovenous dialysis fistulae.

INTRODUCTION: Hemodialysis patients with an arteriovenous fistula can use buttonhole techniques for cannulation. Although buttonholes generally work well, patients may report difficult and painful cannulation, and buttonholes may fail over time. We aimed to assess the effectiveness of tract dilation in treatment of failing buttonholes. METHODS: We retrospectively analyzed data from patients treated with buttonhole tract dilation at an outpatient vascular access center between January 2013 and August 2015. RESULTS: Data from 23 patients were analyzed. There were 51 tract dilation procedures during 36 encounters for failing arteriovenous fistula buttonhole tract(s). The technical success rate for established tract dilation with "blunt-recanalization" was 90% (n = 46). The five remaining buttonholes had "sharp-recanalization" to create and dilate new tract through the buttonhole. For 46 buttonholes treated with "blunt-recanalization," there was an 85% clinical success rate at one week (39 buttonholes), and one was lost to follow-up; there was a 70% clinical success rate after one month (32 buttonholes). In the five buttonholes with "sharp-recanalization," there was only one clinical success with p < 0.05 for difference in success rate compared to "blunt-recanalization" at both one week and one month. There was one complication from "sharp-recanalization" requiring abandonment of the buttonhole tract. DISCUSSION: Buttonhole tract dilation is a useful method to treat difficult cannulation and painful cannulation and has the potential to extend the life of failing buttonholes.

Association of Smoking Status With Mortality and Hospitalization in Hemodialysis Patients.

RATIONALE & OBJECTIVE: The relationship between tobacco smoking and comorbid condition outcomes in hemodialysis (HD) patients is not well understood. This study examined the association of tobacco smoking status with hospitalization and mortality in HD patients. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: Adult HD patients at 2,223 US dialysis centers with HD vintage of 30 days or less who completed a tobacco smoking status survey as part of standard care between April 2013 and June 2015. PREDICTOR: Tobacco smoking category: never smoked, currently living with smoker, former smoker, moderate smoker (<1 pack per day), or heavy smoker (≥1 pack per day). OUTCOMES: Death and hospital admissions within 2 years of the tobacco smoking survey. ANALYTICAL APPROACH: Kaplan-Meier analysis and Cox proportional hazards regression for time to death; cumulative incidence function and Cox proportional hazards regression for time to first hospitalization; negative-binomial regression for number of hospitalizations. RESULTS: Of 22,230 patients studied, 13% were active smokers. Mortality probabilities increased with greater exposure to smoking (17%, 22%, 23%, and 27% for never, moderate, former, and heavy smokers, respectively; P<0.001), as did incidence rates for first hospitalization (23%, 27%, 27%, and 30%, respectively; P<0.001). Compared to never smoked, heavy smokers had the highest mortality rate (HR for heavy smokers, 1.41 [95% CI, 1.18-1.69]; HR for moderate smokers, 1.39 [95% CI, 1.24-1.55]; HR for former smokers, 1.19 [95% CI, 1.11-1.28]). Living with a smoker was not associated with mortality (HR, 0.93; 95% CI, 0.72-1.22). HRs for first hospitalization followed similar patterns. The incidence rate of mortality for active smokers with diabetes was 173.7/1,000 patient-years and 103.5/1,000 patient-years for those who never smoked (incidence rate ratio, 1.68; P<0.001). LIMITATIONS: Self-reported survey without detailed history of smoking/cessation. CONCLUSIONS: Risks for death and hospitalization are elevated among HD patients who smoke, being highest among younger individuals and those with diabetes. Second-hand smoke was not associated with poor clinical outcomes.

Identification and characterization of a calmodulin binding domain in the plasma membrane Ca2+-ATPase from Trypanosoma equiperdum.

The plasma membrane Ca2+-ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC50s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca2+, as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca2+/CaM complex. Moreover, when the peptide was incubated with CaM and Ca2+, a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W1039 and F1056, strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L1042 and W1060 residues might also participate as anchors to form a 1-4-18-22 motif.

Immunohistochemical characterization of tuberculous lesions in sheep naturally infected with Mycobacterium bovis.

BACKGROUND: Sheep have been traditionally considered as less susceptible to Mycobacterium bovis (Mbovis) infection than other domestic ruminants such as cattle and goats. However, there is increasing evidence for the role of this species as a domestic Mbovis reservoir, mostly when sheep share grazing fields with infected cattle and goats. Nevertheless, there is a lack of information about the pathogenesis and the immune response of Mbovis infection in sheep. The goals of this study were to characterize the granuloma stages produced by the natural infection of Mbovis in sheep, to compare them with other species and to identify possible differences in the sheep immune response. Samples from bronchial lymph nodes from twelve Mbovis-naturally infected sheep were used. Four immunohistochemical protocols for the specific detection of T-lymphocytes, B-lymphocytes, plasma cells and macrophages were performed to study the local immune reaction within the granulomas. RESULTS: Differences were observed in the predominant cell type present in each type of granuloma, as well as differences and similarities with the development of tuberculous granulomas in other species. Very low numbers of T-lymphocytes were observed in all granuloma types indicating that specific cellular immune response mediated by T-cells might not be of much importance in sheep in the early stages of infection, when macrophages are the predominant cell type within lesions. Plasma cells and mainly B lymphocytes increased considerably as the granuloma developed being attracted to the lesions in a shift towards a Th2 response against the increasing amounts of mycobacteria. Therefore, we have proposed that the granulomas could be defined as initial, developed and terminal. CONCLUSIONS: Results showed that the study of the lymphoid tissue granulomata reinforces the view that the three different types of granuloma represent stages of lesion progression and suggest an explanation to the higher resistance of sheep based on a higher effective innate immune response to control tuberculosis infection.

Thermal, Structural, and Rheological Characterization of Waxy Starch as a Cryogel for Its Application in Food Processing.

Starch is the major component of cereal, pulses, and root crops. Starch consists of two kinds of glucose polymers, amylose and amylopectin. Waxy starch-with 99⁻100% amylopectin-has distinctive properties, which define its functionality in many food applications. In this research, a novel material was prepared through the cryogelification of waxy starch (WS) using four cycles of freezing and thawing in indirect contact with liquid nitrogen at -150 °C. Polyvinyl alcohol (PVA) was used as a reference. The cryogels were characterized using several validation methodologies: modulated differential scanning calorimetry (MDSC), scanning electron microscopy (SEM), rheology, and Fourier transform infrared (FTIR) spectroscopy with diffuse reflectance (DR). Based on the number of freeze⁻thaw cycles, significant changes were found (P < 0.05) showing important structural modifications as well as reorganization of the polymeric matrix. Two cryogelification cycles of the WS were enough to obtain the best structural and functional characteristics, similar to those of PVA, which has already been tested as a cryogel. From these results, it is concluded that WS has potential as a cryogel for application in food processing.

To cool, or too cool: Is reducing dialysate temperature the optimal approach to preventing intradialytic hypotension?

Abnormal decreases in blood pressure during hemodialysis are frequent in end stage renal disease (ESRD) patients treated with hemodialysis, and thought to be largely due to an inadequate cardiovascular response to the rapid blood volume decline. Intradialytic hypotension (IDH) and cardiac instability during dialysis can increase risks for negative health consequences and is possibly preventable though several types of interventions. One intervention that holds promise for prevention of IDH in hemodialysis patients is to reduce the temperature of the dialysate to or below the patient's core temperature. A considerable number of randomized studies have demonstrated a short term benefit of using a cooler dialysate temperature for the prevention of IDH and improved cardiac stability. Despite this, a key observational study was not able to show long term improvements with lower dialysate temperatures utilized in routine clinical practice, albeit possibly confounded by indication. It appears that cooling the dialysate may be reasonable to consider on an individual basis for patients who suffer from persistent IDH if they can tolerate the adjustment and it is effective. However, careful assessment of the etiology of IDH should be performed when considering treatment options. In this review, we detail the current body of evidence on the effectiveness of using low dialysate temperatures for prevention of IDH in ESRD patients, and suggest areas where further research is needed.

Evidence of the presence of a calmodulin-sensitive plasma membrane Ca2+-ATPase in Trypanosoma equiperdum.

Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca2+ homeostasis. Therefore, the plasma membrane Ca2+-ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif.

Improvements in MBD lab outcomes associated with improved pharmaceutical care in hemodialysis patients.

End stage renal disease (ESRD) patients require a large number of medications and are known to have high rates of nonadherence. It is estimated that >50% of ESRD patients do not take their phosphate binders as prescribed. The renal pharmacy FreseniusRx provides coordinated ESRD medication delivery and adherence support for enrolled patients. We investigated whether coordinated pharmacy care of mineral and bone disorder (MBD) therapies is associated with improvements in laboratory. outcomes. We used data from hemodialysis patients treated at Fresenius Medical Care North America (FMCNA) clinics from February 2014 to January 2015. We included patients who were residing in a state with >100 patients in the FMCNA network, not in a nursing home, and prescribed a phosphate binder and/or calcimimetic. We found 15,287 pharmacy patients who met the study criteria. Concurrent control patients not in the pharmacy were matched to pharmacy patients on a monthly basis that was based off the first date of receipt of therapy from FreseniusRx using 1:1 nearest neighbor matching on the logit of the propensity score for an array of clinical and non-clinical parameters. Logistic regression was used to measure the association between pharmacy care and patients achieving their laboratory goals for phosphorus (PO4) and intact parathyroid hormone (iPTH), and combined goals for total calcium (Ca), PO4, and iPTH. We analyzed data from 30,574 patients (15,287 pharmacy and control). In unadjusted and adjusted analyses, we consistently observed that pharmacy patients were more likely to achieve their MBD laboratory goals as compared to controls. In an adjusted analysis, we found pharmacy patients were more likely to achieve their MBD laboratory targets at 3, 6, 9, and 12 months for PO4 (11.1%, 10.5%, 11.8% and 12.7% respectively), iPTH (8.9%, 17.5%, 23.4% and 27.9% respectively) and combined goals for Ca, PO4, and. iPTH (12.1%, 13.4%, 16.7% and 21.2% respectivelv) versus controls (n<0.01 for all comparisons). These findings indicate that coordinated pharmaceutical care may be associated with improvements in patients achieving their MBD laboratory goals.

ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment.

When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras-dependent effects on spreading and adhesion formation.

The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice.

The lack of current treatment and preventable measures for acute kidney injury (AKI) in hospitalized patients results in an increased mortality rate of up to 80% and elevated health costs. Additionally, if not properly repaired, those who survive AKI may develop fibrosis and long-term kidney damage. The molecular aspects of kidney injury and repair are still uncertain. Hepatocyte growth factor (HGF) promotes recovery of the injured kidney by inducing survival and migration of tubular epithelial cells to repopulate bare tubule areas. HGF-stimulated kidney epithelial cell migration requires the activation of ADP-ribosylation factor 6 (Arf6) and Rac1 via the cytohesin family of Arf-guanine-nucleotide exchange factors (GEFs), in vitro. We used an ischemia and reperfusion injury (IRI) mouse model to analyze the effects of modulating this signaling pathway on kidney recovery. We treated IRI mice with either HGF, the cytohesin inhibitor SecinH3, or a combination of both. As previously reported, HGF treatment promoted rapid improvement of kidney function as evidenced by creatinine (Cre) and blood urea nitrogen (BUN) levels. In contrast, simultaneous treatment with SecinH3 and HGF blocks the ability of HGF to promote kidney recovery. Immunohistochemistry showed that HGF treatment promoted recovery of tubule structure, and had enhanced levels of active, GTP-bound Arf6 and GTP-Rac1. SecinH3 treatment, however, caused a dramatic decrease in GTP-Arf6 and GTP-Rac1 levels when compared to kidney sections from HGF-treated IRI mice. Additionally, SecinH3 counteracted the renal reparative effects of HGF. Our results support the conclusion that cytohesin function is required for HGF-stimulated renal IRI repair.

Wild boar tuberculosis in Iberian Atlantic Spain: a different picture from Mediterranean habitats.

BACKGROUND: Infections with Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex (MTC) are shared between livestock, wildlife and sporadically human beings. Wildlife reservoirs exist worldwide and can interfere with bovine tuberculosis (TB) eradication efforts. The Eurasian wild boar (Sus scrofa) is a MTC maintenance host in Mediterranean Iberia (Spain and Portugal). However, few systematic studies in wild boar have been carried out in Atlantic regions. We describe the prevalence, distribution, pathology and epidemiology of MTC and other mycobacteria from wild boar in Atlantic Spain. A total of 2,067 wild boar were sampled between 2008 and 2012. RESULTS: The results provide insight into the current status of wild boar as MTC and Mycobacterium avium complex (MAC) hosts in temperate regions of continental Europe. The main findings were a low TB prevalence (2.6%), a low proportion of MTC infected wild boar displaying generalized TB lesions (16.7%), and a higher proportion of MAC infections (4.5%). Molecular typing revealed epidemiological links between wild boar and domestic - cattle, sheep and goat - and other wildlife - Eurasian badger (Meles meles) and red fox (Vulpes vulpes) - hosts. CONCLUSIONS: This study shows that the likelihood of MTC excretion by wild boar in Atlantic habitats is much lower than in Mediterranean areas. However, wild boar provide a good indicator of MTC circulation and, given the current re-emergence of animal TB, similar large-scale surveys would be advisable in other Atlantic regions of continental Europe.

Tuberculosis due to Mycobacterium bovis and Mycobacterium caprae in sheep.

Tuberculosis was diagnosed in three flocks of sheep in Galicia, Spain, in 2009 and 2010. Two flocks were infected with Mycobacterium bovis and one flock was infected with Mycobacterium caprae. Infection was confirmed by the comparative intradermal tuberculin test, bacteriology, molecular analysis and histopathology. Sheep have the potential to act as a reservoir for tuberculosis.

Interaction and co-localization of JC virus large T antigen and the F-box protein ß-transducin-repeat containing protein.

Lytic infection and transformation of cultured cells by JC virus (JCV) require five tumor proteins, which interact with factors regulating critical cellular processes. We demonstrate that JCV large T antigen (TAg) binds the F-box proteins ß-transducin-repeat containing protein-1 and 2 (ßTrCP1/2). These interactions involve a phosphodegron (DpSGX(2-4)pS) found in ßTrCP substrates. TAg stability is unaltered, suggesting TAg is a pseudo-substrate. ßTrCP and TAg co-localize in the cytoplasm, and a functional SCF complex is required. We examined whether TAg influences the levels of ß-catenin, a ßTrCP substrate. We were unable to demonstrate that TAg elevates ß-catenin as previously reported, and a mutant TAg unable to bind ßTrCP also had no detectable effect on ß-catenin stability. Results presented in this study link JCV TAg to the cellular degradation complex, SCF(ßTrCP1/2). Proteasomal degradation is essential for proper regulation of cellular functions, and interference with proteasomal pathways highlights possible JCV pathogenic and oncogenic mechanisms.