Regulation of Hfq mRNA and Protein Levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA

Small non-coding RNAs (sRNAs) are important players of gene expression regulation in bacterial pathogens. MtvR is a 136-nucleotide long sRNA previously identified in the human pathogen Burkholderia cenocepacia J2315 and with homologues restricted to bacteria of the Burkholderia cepacia complex. In this work we have investigated the effects of expressing MtvR in Escherichia coli and Pseudomonas aeruginosa. Results are presented showing that MtvR negatively regulates the hfq mRNA levels in both bacterial species. In the case of E. coli, this negative regulation is shown to involve binding of MtvR to the 5′-UTR region of the hfq_Ec mRNA. Results presented also show that expression of MtvR in E. coli and P. aeruginosa originates multiple phenotypes, including reduced resistance to selected stresses, biofilm formation ability, and increased susceptibility to various antibiotics.     

Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells

Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question.     

Growth Factors and Steroid Mediated Regulation of Cytoskeletal Protein Expression in Serum-Deprived Primary Astrocyte Cultures

In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them togheter. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressfull conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Contrasting patterns of mitochondrial and microsatellite genetic structure among Western European populations of tawny owls (Strix aluco)

A recent study of mitochondrial phylogeography of tawny owls (Strix aluco) in western Europe suggested that this species survived the Pleistocene glaciations in three allopatric refugia located in Iberia, Italy, and the Balkans, and the latter was likely the predominant source of postglacial colonization of northern Europe. New data from seven microsatellite loci from 184 individual owls distributed among 14 populations were used to assess the genetic congruence between nuclear and mitochondrial DNA (mtDNA) markers. Microsatellites corroborated the major phylogeographical conclusions reached on the basis of the mtDNA sequences, but also showed important differences leading to novel inferences. Microsatellites corroborated the three major refugia and supported the Balkan origin of northern populations. When corrected for differences in effective population size, microsatellites and mtDNA yielded generally congruent overall estimates of population structure (N*ST=0.12 vs. RST=0.16); however, there was substantial heterogeneity in the RST among the seven nuclear loci that was not correlated with heterozygosity. Populations representing the Balkans postglacial expansion interact with populations from the other two refugia forming two clines near the Alps and the Pyrenees. In both cases, the apparent position of the contact zones differed substantially between markers due to the genetic composition of populations sampled in northern Italy and Madrid. Microsatellite data did not corroborate the lower genetic diversity of northern, recently populated regions as was found with mtDNA; this discrepancy was taken as evidence for a recent bottleneck recovery. Finally, this study suggests that congruence among genetic markers should be more likely in cases of range expansion into new areas than when populations interact across contact zones.     

Usefulness of 1,3 Beta-<Emphasis Type="SmallCaps">d</Emphasis>-Glucan Detection in non-HIV Immunocompromised Mechanical Ventilated Critically Ill Patients with ARDS and Suspected <Emphasis Type="Italic">Pneumocystis jirovecii</Emphasis> Pneumonia

Introduction

Pneumocystis jirovecii pneumonia (PCP) is a major cause of disease in immunocompromised individuals. Diagnosis is typically obtained by microscopy and/or PCR. For ambiguous PCR results, we evaluated the new biomarker 1,3-Beta-d-Glucan (BDG).

Methods

BDG serum levels were assessed and correlated to PCR results in immunosuppressed patients with ARDS.

Results

11 (22%) out of 50 patients had suspected PCP. APACHE II (26 vs. 24; p < 0.002), SOFA score (16 vs. 14; p < 0.010) and mortality rate (34 vs. 69% p < 0.004; 34 vs. 80% p < 0.003) were significantly altered in patients with positive (pPCR) and slightly positive (spPCR) PCJ PCR as compared to patients with no-PCP (nPCP). BDG levels were significantly lower in patients with nPCP (86; 30–315 pg/ml) than in patients with pPCR (589; 356–1000 pg/ml; p < 0.001) and spPCP (398; 297–516 pg/ml; p < 0.004) referring to the cutoff in this study for PCP of 275 pg/ml. An overall sensitivity (S) of 92% (95% CI 86–96%) and specificity (SP) of 84% (95% CI 79–85%) for PCP were found for the BDG Fungitell assay. In detail, S of 98% (95% CI 94–100%) and SP of 86% (95% CI 82–92%) for pPCP and S of 98% (95% CI 96–100%) and SP of 88% (95% CI 86–96%) for spPCO were found.

Conclusion

Serum BDG levels were strongly elevated in PCP, and the negative predictive value is high. BDG could be used as a preliminary test for patients with suspected PCP, especially in patients with slightly positive PCR results.

     

Chemical Composition of Essential Oils of Xanthium spinosum L., an Invasive Species of Corsica

Xanthium spinosum L. is a highly invasive plant originated from South America throughout the world as well as in Corsica Island. The chemical composition of X. spinosum essential oils from 25 Corsican locations was investigated using GC‐FID and GC/MS. Seventy‐four components, which accounted for 96.2% of the total amount, were reported for the first time in the essential oil from aerial parts. The main compounds were eudesma‐4(14),7‐dien‐1β‐ol ( 61 ; 21.3%), germacrene D ( 36 ; 8.8%) and cadalene ( 60 ; 8.7%). Comparison with the literature highlighted the originality of the Corsican essential oil and eudesma‐4(14),7‐dien‐1β‐ol could be used as taxonomical marker to the systematics of the Xanthium genus. The essential oils obtained from separate organs and during the plant vegetative cycle were also studied to gain more knowledge about the correlations between the volatile production and the phenological states of this weed. The production of oxygenated sesquiterpenes was predominant during the plant‐flowering process. The study focuses on direct correlation between the chemical composition of individual 25 oil samples and the morphological differences of the plant. Our results have gained more knowledge about the secondary metabolite production that occurs during the plant life, they could be interesting in order to manage the dispersal of X. spinosum.     

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.     

The change of behaviour of two strains of Leishmania after cultivation in a defined medium

Attempts have been made to characterize two strains of Leishmania that became infective to golden hamsters only after they had been maintained for several years in a chemically defined culture medium. Observations were made on the growth rates of promastigotes in vitro, course of infection in hamsters, morphology of amastigotes, and electrophoretic mobility patterns of eight isoenzymes. Information was obtained about the buoyant densities of n-DNA and k-DNA, and one strain was tested against monoclonal antibodies. The identity of both strains remains obscure.     

Use of the paired samples (cerebrospinal fluid and serum) in immunodiagnostic of active and inactive human neurocysticercosis

Paired samples of cerebrospinal fluid (CSF) and serum of 30 patients--10 with active, 10 with inactive neurocysticercosis (NCC), and 10 control subjects--were evaluated by enzyme-linked immunosorbent assay (ELISA) using two Taenia crassiceps metacestode extracts as antigen in order to detect IgG antibodies. In active NCC, high levels of IgG were detected (p < 0.05). The CSF samples showed 80% (CI 72-88) of reactivity in the saline extract (S) and 90% (CI 84-95) in sodium dodecyl sulphate (SDS) and the serum samples were reactive in 90% (CI 84-95) and 100% (CI 98-100) in the S and SDS antigenic extracts, respectively. The use of the paired samples of CSF and serum in active NCC showed equivalent results suggesting that the serum samples could be used as a screening in those patients whose CSF puncture is counter-indicated.     

Activation of NMDA receptors induces protein kinase A-mediated phosphorylation and degradation of matrin 3. Blocking these effects prevents NMDA-induced neuronal death

Activation of NMDA receptors leads to activation of cAMP-dependent protein kinase (PKA). The main substrates phosphorylated by PKA following NMDA receptor activation remain unidentified. The aim of this work was to identify a major substrate phosphorylated by PKA following NMDA receptor activation in cerebellar neurones in culture, and to assess whether this phosphorylation may be involved in neuronal death induced by excessive NMDA receptor activation. The main PKA substrate following NMDA receptor activation was identified by MALDI-TOFF fingerprinting as the nuclear protein, matrin 3. PKA-mediated phosphorylation of matrin 3 is followed by its degradation. NMDA receptor activation in rat brain in vivo by ammonia injection also induced PKA-mediated matrin 3 phosphorylation and degradation in brain cell nuclei. Blocking NMDA receptors in brain in vivo with MK-801 reduced basal phosphorylation of matrin 3, suggesting that it is modulated by NMDA receptors. Inhibition of PKA with H-89 prevents NMDA-induced phosphorylation and degradation of matrin 3 as well as neuronal death. These results suggest that PKA-mediated phosphorylation of matrin 3 may serve as a rapid way of transferring information from synapses containing NMDA receptors to neuronal nuclei under physiological conditions, and may contribute to neuronal death under pathological conditions.