Highly Sensitive,Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.     

Serum Metalloproteinases 2 and 9 as Predictors of Gait Status,Pressure Ulcer and Mortality after Hip Fracture

Introduction

The aim of this study is to evaluate the serum activity of metalloproteinases (MMPs) -2 and -9 as predictors of pressure ulcer (PU), gait status and mortality 6 months after hip fracture.

Methods

Eighty-seven patients over the age of 65 admitted to the orthopedic unit from January to December 2010 with hip fracture were prospectively evaluated. Upon admission, patient demographic information, including age, gender and concomitant diseases, was recorded. Blood samples were taken for analysis of MMP -2 and -9 activity by gel zymography and for biochemical examination within the first 72 hours of the patient’s admission, after clinical stabilization. The fracture pattern (neck, trochanteric or subtrochanteric), time from admission to surgery, surgery duration and length of hospital stay were also recorded.

Results

Two patients were excluded due to the presence of pathological fractures (related to cancer), and three patients were excluded due to the presence of PU before admission. Eighty-two patients, with a mean age of 80.4 ± 7.3 years, were included in the analysis. Among these patients, 75.6% were female, 59.8% had PU, and 13.4% died 6 months after hip fracture. All patients underwent hip fracture repair. In a univariate analysis, there were no differences in serum MMP activity between hip fracture patients with or without PU. In addition, the multiple logistic regression analysis models, which were adjusted by age, gender, length of hospital stay and C-reactive protein, showed that the pro-MMP-9 complexed with neutrophil gelatinase-associated lipocalin form (130 kDa) was associated with gait status recovery 6 months after hip fracture.

Conclusions

In conclusion, serum pro-MMP-9 is a predictor of gait status recovery 6 months after hip fracture.     

Incomplete Normalization of Regulatory T-Cell Frequency in the Gut Mucosa of Colombian HIV-Infected Patients Receiving Long-Term Antiretroviral Treatment

Introduction

To evaluate the effect of late initiation of HAART and poor immune reconstitution on the frequency of regulatory T-cells (Treg) in the peripheral blood and gut of HIV-infected patients, we studied Colombian HIV-infected patients who had been on suppressive HAART for at least one year. They had undetectable viremia but were either immunological responders (HIR); (CD4 counts >500 cells/µl) or non-immunological responders (NIR); (CD4 T-cell count <300 cells/µl). Untreated HIV-infected patients and uninfected controls from the same region were also evaluated.

Methods

Frequency and phenotype of regulatory T-cells (Treg) were analyzed in gut biopsies and blood samples. The functional effect of Treg depletion on CMV and HIV responses was determined. Markers of immune activation and circulating LPS levels were quantified.

Results

Untreated patients exhibited high Treg frequency in PBMC and gut, and their Treg express high levels of CTLA-4 and PD-1. Although HAART significantly decreased mucosal Treg frequency, it did not normalize it in any of the treated groups (HIR and NIR patients). Treg normalization was observed in the blood of HIR patients following HAART, but did not occur in NIR patients. Treg from HIV-infected patients (treated or not) suppressed HIV and hCMV-specific T-cells from gut and blood. Plasma LPS levels and percentage of HLA-DR+CD38+ T-cells were significantly elevated in all infected groups compared to controls.

Conclusions

These findings suggest that control of viral replication is not sufficient to normalize gut Treg frequency in patients, independent of their response to HAART. Furthermore, persistence of functional Treg in the gut appears to be associated with the failure of HAART to repair mucosal damage.     

Association of Pro-Inflammatory Cytokines and Iron Regulatory Protein 2 (IRP2) with Leishmania Burden in Canine Visceral Leishmaniasis

Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2) were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.     

Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion.     

Phylogeographic Pattern and Extensive Mitochondrial DNA Divergence Disclose a Species Complex within the Chagas Disease Vector Triatoma dimidiata

Background

Triatoma dimidiata is among the main vectors of Chagas disease in Latin America. However, and despite important advances, there is no consensus about the taxonomic status of phenotypically divergent T. dimidiata populations, which in most recent papers are regarded as subspecies.

Methodology and Findings

A total of 126 cyt b sequences (621 bp long) were produced for specimens from across the species range. Forty-seven selected specimens representing the main cyt b clades observed (after a preliminary phylogenetic analysis) were also sequenced for an ND4 fragment (554 bp long) and concatenated with their respective cyt b sequences to produce a combined data set totalling 1175 bp/individual. Bayesian and Maximum-Likelihood phylogenetic analyses of both data sets (cyt b, and cyt b+ND4) disclosed four strongly divergent (all pairwise Kimura 2-parameter distances >0.08), monophyletic groups: Group I occurs from Southern Mexico through Central America into Colombia, with Ecuadorian specimens resembling Nicaraguan material; Group II includes samples from Western-Southwestern Mexico; Group III comprises specimens from the Yucatán peninsula; and Group IV consists of sylvatic samples from Belize. The closely-related, yet formally recognized species T. hegneri from the island of Cozumel falls within the divergence range of the T. dimidiata populations studied.

Conclusions

We propose that Groups I–IV, as well as T. hegneri, should be regarded as separate species. In the Petén of Guatemala, representatives of Groups I, II, and III occur in sympatry; the absence of haplotypes with intermediate genetic distances, as shown by multimodal mismatch distribution plots, clearly indicates that reproductive barriers actively promote within-group cohesion. Some sylvatic specimens from Belize belong to a different species – likely the basal lineage of the T. dimidiata complex, originated ∼8.25 Mya. The evidence presented here strongly supports the proposition that T. dimidiata is a complex of five cryptic species (Groups I–IV plus T. hegneri) that play different roles as vectors of Chagas disease in the region.     

Thioredoxin h2 contributes to the redox regulation of mitochondrial photorespiratory metabolism

Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O₂ conditions. However, an increase in the stoichiometry of photorespiratory CO₂ release was found during O₂-dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD⁺ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.     

The threat of emerging and re-emerging pathogenic Sporothrix species

Mycopathologia - Sporotrichosis is a neglected subcutaneous mycosis of humans and animals acquired by traumatic inoculation of soil and plant material (classical route) contaminated with infectious...     

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Molecular Biology Reports - Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic...     

Co-Inheritance of alpha-thalassemia and sickle cell disease in a cohort of Angolan pediatric patients

Molecular Biology Reports - The aim of this study was to explore the association between alpha-thalassemia, fetal hemoglobin, hematological indices, and clinical adverse events in Angolan sickle...