Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains

The rhizobia-legume symbiosis requires a coordinated molecular interaction between the symbionts, initiated by seed and root exudation of several compounds, mainly flavonoids, that trigger the expression of nodulation genes in the bacteria. Since the role of flavonoids seems to be broader than the induction of nodulation genes, we aimed at characterizing genistein-induced proteins of Bradyrhizobium japonicum CPAC 15 (= SEMIA 5079), used in commercial soybean inoculants in Brazil, and of two genetically related strains grown in vitro. Whole-cell proteins were extracted both from induced (1 μM genistein) and from non-induced cultures of the three strains, and separated by two-dimensional electrophoresis. Spot profiles were compared between the two conditions and selected spots were excised and identified by mass spectrometry. Forty-seven proteins were significantly induced by genistein, including several hypothetical proteins, the cytoplasmic flagellar component FliG, periplasmic ABC transporters, a protein related to biosynthesis of exopolysaccharides (ExoN), and proteins involved in redox-state maintenance. Noteworthy was the induction of the PhyR-σ^EcfG regulon, recently demonstrated to be involved in the symbiotic efficiency of, and general stress response in B. japonicum. Our results confirm that the role of flavonoids, such as genistein, can go far beyond the expression of nodulation-related proteins in B. japonicum.     

Analysis of fungal diversity in <Emphasis Type="Italic">Orchis tridentata</Emphasis> Scopoli

We have assessed the identities of fungi associated with Orchis tridentata, an endangered orchid species growing in open woodland and poor grassland of Central and Southern Europe. Fungal diversity in ten O. tridentata adult individuals collected in two protected areas of Central Italy was analysed by means of morphological and molecular methods. Sequencing of the cloned ITS fungal inserts corresponding to the dominant PCR products obtained from amplification of total root DNA with ITS1F and ITS4 primers revealed a variety of fungal species occurring in O. tridentata roots. Among them, members of the basidiomycete families Ceratobasidiaceae, Tulasnellaceae and Hymenogastraceae were recovered, together with ascomycetes belonging to Leptodontidium and Terfezia. The implications of these results in the understanding of O. tridentata biology and for the conservation of this threatened orchid species are discussed.     

Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity

Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles.     

Impact of natalizumab on cognitive performances and fatigue in relapsing multiple sclerosis: a prospective, open-label, two years observational study

Background and Objectives

Natalizumab reduces the relapse rate and magnetic resonance imaging activity in patients with Relapsing-Remitting Multiple Sclerosis (RRMS). So far the influence of natalizumab on cognitive functions and fatigue in MS remains uncertain. The aim of this prospective, open-label, observational study was to evaluate the possible effects of natalizumab on cognition and fatigue measures in RRMS patients treated for up to two years.

Methods

Cognitive performances were examined by the Rao''s Brief Repeatable Battery (BRB), the Stroop test (ST) and the Cognitive Impairment Index (CII), every 12 months. Patients who failed in at least 3 tests of the BRB and the ST were classified as cognitively impaired (CI). Fatigue Severity Scale (FSS) was administered every 12 months to assess patient''s self-reported fatigue. One hundred and 53 patients completed 1 and 2 year-natalizumab treatment, respectively.

Results

After 1 year of treatment the percentage of CI patients decreased from 29% (29/100) at baseline to 19% (19/100) (p = 0.031) and the mean baseline values of CII (13.52±6.85) and FSS (4.01±1.63) scores were significantly reduced (10.48±7.12, p<0.0001 and 3.61±1.56, p = 0.008). These significant effects were confirmed in the subgroup of patients treated up to two years.

Conclusions

These results demonstrate that a short-term NTZ treatment may significantly improve cognitive performances and fatigue in RRMS patients.     

Low Parasite Load Estimated by qPCR in a Cohort of Children Living in Urban Area Endemic for Visceral Leishmaniasis in Brazil

Background

An important issue associated with the control of visceral leishmaniasis is the need to identify and understand the relevance of asymptomatic infection caused by Leishmania infantum. The aim of this study was to follow the course of asymptomatic L. infantum infection in children in an area of Brazil where it is endemic. The children were assessed twice during a 12-month period.

Methodology

In this population study, 1875 children, ranging from 6 months to 7 years of age, were assessed. Blood samples were collected on filter papers via finger prick and tested by ELISA (L. infantum soluble antigen and rk39). Seropositives samples (n = 317) and a number of seronegatives samples (n = 242) were subjected to qPCR. After 12 months, blood samples were collected from a subgroup of 199 children and tested for Leishmania spp. to follow the course of infection.

Principal Findings

At baseline qPCR testing identified 82 positive samples. The prevalence rate, as estimated for 1875 children based on the qPCR results, was 13.9%. The qPCR testing of whole blood samples collected from a cohort of children after 12 months (n = 199) yielded the following results: of the 44 (22.1%) children with positive qPCR results at baseline, only 10 (5.0%) remained positive, and 34 (17.1%) became negative; and of the 155 (77.9%) children with negative qPCR results, 131 (65.8%) remained negative, and 24 (12.1%) became positive at the follow-up measurement. The samples with positive findings at baseline (n = 82) had a mean of 56.5 parasites/mL of blood; and at follow-up the mean positive result was 7.8 parasites/mL.

Conclusions

The peripheral blood of asymptomatic children had a low and fluctuating quantity of Leishmania DNA and a significant decrease in parasitemia at 1-year follow-up. Quantitative PCR enables adequate monitoring of Leishmania infection.     

Biomarkers of Good EULAR Response to the B Cell Depletion Therapy in All Seropositive Rheumatoid Arthritis Patients: Clues for the Pathogenesis

Objective

To find out whether a high number of auto-antibodies can increase the probability of a “good-EULAR response” and to identify the possible biomarkers of response in seropositive rheumatoid arthritis (RA) patients undergoing the B cell depletion therapy (BCDT).

Patients and Methods

One hundred and thirty-eight patients with long standing RA (LSRA), 75% non or poorly responsive to one or more TNFα blockers, all seropositive for at least one autoantibody (AAB) (RF-IgM, RF-IgA, RF-IgG, anti-MCV, ACPA-IgG, ACPA-IgA, ACPA-IgM) received one full course of BCDT. The major outcomes (moderate or good-EULAR response) were assessed after 6 months of therapy. The IL6 and BAFF levels were also determined.

Results

At a 6-month follow-up, 33 (23.9%) of the RA patients achieved a good EULAR response. Having up to 5-AABs positivity increased the chances for treatment response. After a logistic regression analysis, however, only 4 baseline factors arose as associated with a good-EULAR response: no steroid therapy (OR = 6.25), a lymphocyte count <1875/uL (OR = 10.74), a RF-IgG level >52.1 IU/ml (OR = 8.37) and BAFF levels <1011 pg/ml (OR = 7.38). When all the AABs, except for RF-IgM and ACPA-IgG, were left in the analysis, the two final predictors were no-steroid therapy and low lymphocyte count.

Discussion

The number of AABs increased the chances of being a “good-EULAR” responder. The only predictors, however, at the baseline of a good response in this seropositive cohort of RA patients were 2 simple variables – no steroids and lymphocyte count – and two laboratory assays – IgG-RF and BAFF.     

Proteomic profiling of Rhizobium tropici PRF 81: Identification of conserved and specific responses to heat stress

ABSTRACT: BACKGROUND: Rhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28degreesC and 35degreesC. RESULTS: Two-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses. CONCLUSIONS: Our data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species.     

Immunosuppression in cardiac graft rejection: a human in vitro model to study the potential use of new immunomodulatory drugs

CXCL10-CXCR3 axis plays a pivotal role in cardiac allograft rejection, so that targeting CXCL10 without inducing generalized immunosuppression may be of therapeutic significance in allotransplantation. Since the role of resident cells in cardiac rejection is still unclear, we aimed to establish reliable human cardiomyocyte cultures to investigate Th1 cytokine-mediated response in allograft rejection. We used human fetal cardiomyocytes (Hfcm) isolated from fetal hearts, obtained after legal abortions. Hfcm expressed specific cardiac lineage markers, specific cardiac structural proteins, typical cardiac currents and generated ventricular action potentials. Thus, Hfcm represent a reliable in vitro tool for allograft rejection research, since they resemble the features of mature cells. Hfcm secreted CXCL10 in response to IFNgamma and TNFalphaalpha; this effect was magnified by cytokine combination. Cytokine synergy was associated to a significant TNFalpha-induced up-regulation of IFNgammaR. The response of Hfcm to some currently used immunosuppressive drugs compared to rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist and Th1-mediated response inhibitor, was also evaluated. Only micophenolic acid and rosiglitazone halved CXCL10 secretion by Hfcm. Given the pivotal role of IFNgamma-induced chemokines in Th1-mediated allograft rejection, these preliminary results suggest that the combined effects of immunosuppressive agents and rosiglitazone could be potentially beneficial to patients receiving heart transplants.     

Increased expression of the neuropeptide Y receptor Y1 gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABA(A) receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y(1)R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y(1) receptor for NPY linked to the lacZ gene, we previously showed that long-term treatment with benzodiazepine receptor ligands modulates Y(1) receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y(1)R/LacZ transgene expression, as determined by quantitative histochemical analysis of beta-galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and beta-galactosidase expression in the medial amygdala. Finasteride, a 5alpha-reductase inhibitor, prevented both of these effects. Long-term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected beta-galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y(1) receptor gene expression through modulation of GABA(A) receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.