Structural and biochemical characterization of the M405S variant of Desulfovibrio vulgaris formate dehydrogenase.

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO2 reduction, together with an increased sensitivity to oxygen inactivation.

Mozambique public investment in the water and sanitation sector and the targets of SDG6.

Many developing countries may not reach the targets of providing access to safe water sources and sanitation services for all by 2030. Census data from Mozambique show that the country's population is one of fastest growing in the Sub-Saharan region. Between the 2007 and 2017 censuses more than seven million people were added to the total population. By 2030 about 11 million people will be added to Mozambique's population. This will pose a huge challenge for the water and sanitation services. Access to these services is a fundamental requirement for the wellbeing of individuals and the development of nations. The last data from a Survey on Mozambique Family Budgets (IOF 2019/20), show that 55.7% of a total population has access to safe water sources. In contrast, access to sanitation services (31% of the population), has not kept pace with the progress made in water access. In this study, based on data from the General State Account of Mozambique, which includes the description of the annual investment made by the government and using the results of the Family Budget Surveys, it can be seen that if the average percentage values of public investment of 2009 to 2021 are the same in the following years as regards the water and sanitation sector, Mozambique will not reach the Sustainable Development Goals 6 targets in 2030.

Implementation of a Zebrafish Health Program in a Research Facility: A 4-Year Retrospective Study.

In the past two decades, zebrafish (Danio rerio)-based research has contributed to significant scientific advances. Still, husbandry and health programs did not evolve at the same pace, as evidenced by the absence of general guidelines. Health monitoring is essential to animal welfare, to permit animal exchanges across facilities, to contribute to robust experimental results, and for data reproducibility. In this study, we report a health program implemented in a zebrafish research facility to prevent, monitor, and control pathogen, and disease dissemination. This program includes quarantine, routine health screening of sentinels, and nonroutine screenings of retired animals and sick/moribund individuals. An extensive list of clinical signs, lesions, and pathogens was monitored based on: daily observation of fish, necropsy, histology, and bacterial culture. The results indicate that the combined analysis of sentinels with the evaluation of sick/moribund animals enables a comprehensive description not only of pathogen prevalence but also of clinical and histopathologic lesions of resident animals. The establishment of a quarantine program revealed to be effective in the reduction of Pseudoloma neurophilia frequency in the main aquaria room. Finally, characterization of the colony health status based on this multiapproach program shows a low prevalence of lesions and pathogens in the facility.

Specific Mechanisms Underlying Right Heart Failure: The Missing Upregulation of Superoxide Dismutase-2 and Its Decisive Role in Antioxidative Defense.

AIMS: Research into right ventricular (RV) physiology and identification of pathomechanisms underlying RV failure have been neglected for many years, because function of the RV is often considered less important for overall hemodynamics and maintenance of blood circulation. In view of this, this study focuses on identifying specific adaptive mechanisms of the RV and left ventricle (LV) during a state of chronic nitric oxide (NO) deficiency, one of the main causes of cardiac failure. NO deficiency was induced in rats by L-NAME feeding over a 4 week period. The cardiac remodeling was then characterized separately for the RV/LV using quantitative real-time polymerase chain reaction, histology, and functional measurements. RESULTS: Only the RV underwent remodeling that corresponded morphologically and functionally with the pattern of dilated cardiomyopathy. Symptoms in the LV were subtle and consisted primarily of moderate hypertrophy. A massive increase in reactive oxygen species (ROS) (+4.5±0.8-fold, vs. control) and a higher degree of oxidized tropomyosin (+46%±4% vs. control) and peroxynitrite (+32%±2% vs. control) could be identified as the cause of both RV fibrosis and contractile dysfunction. The expression of superoxide dismutase-2 was specifically increased in the LV by 51%±3% and prevented the ROS increase and the corresponding structural and functional remodeling. INNOVATION: This study identified the inability of the RV to increase its antioxidant capacity as an important risk factor for developing RV failure. CONCLUSION: Unlike the LV, the RV did not display the necessary adaptive mechanisms to cope with increased oxidative stress during a state of chronic NO deficiency.

Phenolic profile of Douro wines and evaluation of their NO scavenging capacity in LPS-stimulated RAW 264.7 macrophages.

The phenolic profile of two samples of commercial Douro red wines, a table red wine (RM) and a Port wine (PR), was analysed by HPLC-DAD after purification by solid phase extraction (SPE). Eighteen phenolic compounds belonging to benzoic acids, hydroxycinnamic acids and anthocyanins were determined. The presence of tannins in both wines and of polymeric anthocyanins in Port wine could be inferred by the chromatograms registered at 280 and 500 nm, respectively. The wine extracts showed capacity to scavenge sodium nitroprusside-released nitric oxide (NO), RM being more effective than PR, which can be partly attributed to its higher content in monomeric anthocyanins. Using a model of macrophages (RAW 264.7 cells) stimulated with bacterial lipopolysaccharide (LPS) the samples showed a similar potential, which may indicate the importance of polymeric anthocyanins and the higher amount of phenolic acids present in PR to decrease cells NO levels.

Structural and functional concepts in current mouse phenotyping and archiving facilities.

Collecting and analyzing available information on the building plans, concepts, and workflow from existing animal facilities is an essential prerequisite for most centers that are planning and designing the construction of a new animal experimental research unit. Here, we have collected and analyzed such information in the context of the European project Infrafrontier, which aims to develop a common European infrastructure for high-throughput systemic phenotyping, archiving, and dissemination of mouse models. A team of experts visited 9 research facilities and 3 commercial breeders in Europe, Canada, the United States, and Singapore. During the visits, detailed data of each facility were collected and subsequently represented in standardized floor plans and descriptive tables. These data showed that because the local needs of scientists and their projects, property issues, and national and regional laws require very specific solutions, a common strategy for the construction of such facilities does not exist. However, several basic concepts were apparent that can be described by standardized floor plans showing the principle functional units and their interconnection. Here, we provide detailed information of how individual facilities addressed their specific needs by using different concepts of connecting the principle units. Our analysis likely will be valuable to research centers that are planning to design new mouse phenotyping and archiving facilities.

Steroid treatments in mice do not alter the number and function of regulatory T cells, but amplify cyclophosphamide-induced autoimmune disease.

Corticosteroids are commonly used in the therapy of autoimmune disease (AID), although they are rarely, if ever, curative. This failure may result from their deleterious effects on regulatory T cells (Treg). In this work, we directly tested the effects of hydrocortisone (HC) administration on Treg number and function in established mouse models of multiple sclerosis and colitis. Treatment with pertussis toxin (Ptx) or Cyclophosphamide (Cyp), two compounds known to affect Treg function served as controls. We first show that contrarily to Ptx, HC administration to mice transgenic for a TCR specific to myelin basic protein induces a mild lymphopenia, without selective depletion of Treg, nor induction of experimental autoimmune encephalomyelitis (EAE). We next report that HC administration to normal mice has no effect on Treg suppressive function tested in vitro. Moreover, we document that Treg isolated from HC-treated animals maintain their capacity to prevent T cell-induced colitis. In contrast, the combined administration of HC and Cyp, as is frequently used in the therapy of severe AID, dramatically enhanced the deleterious effect of Cyp on Treg number and function. Our analysis indicates that while a short course of corticosteroids alone is not deleterious to immune regulation, combined therapies, notably with Cyp, should be avoided.

Notch1 engagement by Delta-like-1 promotes differentiation of B lymphocytes to antibody-secreting cells.

Notch signaling regulates B and T lymphocyte development and T cell effector class decision. In this work, we tested whether Notch activity affects mature B cell activation and differentiation to antibody-secreting cells (ASC). We show increased frequency of ASC in cultures of splenic B cells activated with LPS or anti-CD40 when provided exogenous Notch ligand Delta-like-1 (Dll1). Our results indicate that Notch-Dll1 interaction releases a default pathway that otherwise inhibits Ig secretion upon B cell activation. Thus, Dll1 enhanced spontaneous Ig secretion by naturally activated marginal zone B and B1 cells and reversed the inhibition of ASC differentiation mediated by B cell receptor crosslinking during LPS. Moreover, suppression of Notch signaling in B cell expression of either a dominant-negative mutant form of Mastermind-like 1 or a null mutation of Notch1 not only prevented Dll1-mediated enhancement of ASC differentiation but also reduced dramatically LPS-induced Ig secretion. Finally, we show that Dll1 and Jagged-1 are differentially expressed in discrete areas of the spleen, and that the effect of Notch engagement on Ig secretion is ligand-specific. These results indicate that Notch ligands participate in the definition of the mature B cell microenvironment that influences their terminal differentiation.

Exposure to mixed asymptomatic infections with Trypanosoma cruzi, Leishmania braziliensis and Leishmania chagasi in the human population of the greater Amazon.

Lack of conservation of the Amazon tropical rainforest has imposed severe threats to its human population living in newly settled villages, resulting in outbreaks of some infectious diseases. We conducted a seroepidemiological survey of 1100 inhabitants of 15 villages of Paço do Lumiar County, Brazil. Thirty-five (3%) individuals had been exposed to Trypanosoma cruzi (Tc), 41 (4%) to Leishmania braziliensis (Lb) and 50 (4.5%) to Leishmania chagasi (Lc) infections. Also, 35 cases had antibodies that were cross-reactive against the heterologous kinetoplastid antigens. Amongst these, the Western blot assays revealed that 11 (1%) had Tc and Lb, that seven (0.6%) had Lc and Tc, and that 17 (1.6%) had Lb and Lc infections. All of these cases of exposures to mixed infections with Leishmania sp, and eight of 11 cases of Tc and Lb were confirmed by specific PCR assays and Southern hybridizations. Two cases had triple infections. We consider these asymptomatic cases showing phenotype and genotype markers consistent with mixed infections by two or more kinetoplastid flagellates a high risk factor for association with Psychodidae and Triatominae vectors blood feeding and transmitting these protozoa infections. This is the first publication showing human exposure to mixed asymptomatic kinetoplastid infections in the Amazon.

Foxp3+ CD25- CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion.

Expression of the IL-2 receptor alpha chain (CD25) by peripheral CD4 T cells follows cellular activation. However, CD25 expression by CD4 cells is widely used as a marker to identify regulatory T cells (T(R)), although cells with regulatory properties are also found in the CD4+CD25- subset. By using in vivo functional assays and Foxp3 expression as a faithful marker of T(R) differentiation, we have evaluated the requirements for CD25 expression by peripheral T(R). We first show that in vivo depletion of CD25+ cells prevents the development of spontaneous encephalomyelitis in recombination-activating gene (RAG)-deficient anti-myelin basic protein T cell antigen receptor (TCR) transgenic mice, and allows disease induction in otherwise healthy RAG-competent transgenic mice. Similar treatment in normal thymectomized animals is followed by the fast recovery of a normal number of CD25+ T(R). Consistently, Foxp3-expressing T(R) encompassed in the CD25- cell population convert to CD25+ after homeostatic expansion and are selectable by IL-2 in vitro. Surface expression of CD25 on T(R) is controlled by the activity of conventional CD4 cells and is fully labile because it can be lost and regained without affecting the functional potential of the cells. These findings reveal that Foxp3-expressing CD25- cells constitute a peripheral reservoir of differentiated T(R), recruited to the CD25+ pool upon homeostatic expansion and/or activation. This analysis, together with the notion that physiological commitment of T(R) takes place exclusively in the thymus should help for the interpretation of experiments assessing peripheral T(R) differentiation from naive CD4 T cells, defined as CD25-.