In silico evaluation of pharmacokinetics properties of withanolides and simulation of their biological activities against Alzheimer's disease.

The withanolides are naturally occurring steroidal lactones found mainly in plants of the Solanaceae family. The subtribe Withaninae includes species like Withania sominifera, which are a source of many bioactive withanolides. In this work, we selected and evaluate the ADMET-related properties of 91 withanolides found in species of the subtribe Withaninae computationally, to predict the relationship between their structures and their pharmacokinetic profiles. We also evaluated the interaction of these withanolides with known targets of Alzheimer's disease (AD) through molecular docking and molecular dynamics. Withanolides presented favorable pharmacokinetic properties, like high gastrointestinal absorption, lipophilicity (logP ≤ 5), good distribution and excretion parameters, and a favorable toxicity profile. The specie Withania aristata stood out as an interesting source of the promising withanolides classified as 5-ene with 16-ene or 17-ene. These withanolides presented a favourable pharmacokinetic profile and were also highlighted as the best candidates for inhibition of AD-related targets. Our results also suggest that withanolides are likely to act as cholinesterase inhibitors by interacting with the catalytic pocket in an energy favorable and stable way.Communicated by Ramaswamy H. Sarma.

Can the post-ruminal urea release impact liver metabolism, and nutritional status of beef cows at late gestation?

We aimed to evaluate the effects of post-ruminal supply of urea (PRU) on nutritional status, and liver metabolism of pregnant beef cows during late gestation. Twenty-four Brahman dams, pregnant from a single sire, and weighing 545 kg ± 23 kg were confined into individual pens at 174 ± 23 d of gestation, and randomly assigned into one of two dietary treatments up to 270 d of gestation: Control (CON, n = 12), consisting of a basal diet supplemented with conventional urea, where the cows were fed with diets containing 13.5 g conventional urea per kg dry matter; and PRU (PRU, n = 12), consisting of a basal diet supplemented with a urea coated to extensively prevent ruminal degradation while being intestinally digestible, where the cows were fed with diets containing 14,8 g urea protected from ruminal degradation per kg dry matter. Post-ruminal supply of urea reduced the urine levels of 3-methylhistidine (P = 0.02). There were no differences between treatments for dry matter intake (DMI; P = 0.76), total digestible nutrient (TDN) intake (P = 0.30), and in the body composition variables, such as, subcutaneous fat thickness (SFT; P = 0.72), and rib eye area (REA; P = 0.85). In addition, there were no differences between treatments for serum levels of glucose (P = 0.87), and serum levels of glucogenic (P = 0.28), ketogenic (P = 0.72), glucogenic, and ketogenic (P = 0.45) amino acids, neither for urea in urine (P = 0.51) as well as urea serum (P = 0.30). One the other hand, enriched pathways were differentiated related to carbohydrate digestion, and absorption, glycolysis, pyruvate metabolism, oxidative phosphorylation, pentose phosphate pathway, and biosynthesis of amino acids of the exclusively expressed proteins in PRU cows. Shifting urea supply from the rumen to post-ruminal compartments decreases muscle catabolism in cows during late gestation. Our findings indicate that post-ruminal urea supplementation for beef cows at late gestation may improve the energy metabolism to support maternal demands. In addition, the post-ruminal urea release seems to be able to trigger pathways to counterbalance the oxidative stress associated to the increase liver metabolic rate.

Influence of glucose on xylose metabolization by Spathaspora passalidarum.

The yeast Spathaspora passalidarum is able to produce ethanol from D-xylose and D-glucose. However, it is not clear how xylose metabolism is affected by D-glucose when both sugars are available in the culture medium. The aims of this work were to evaluate the influence of D-glucose on D-xylose consumption, ethanol production, gene expression, and the activity of key xylose-metabolism enzymes under both aerobic and oxygen-limited conditions. Ethanol yields and productivities were increased in culture media containing D-xylose as the sole carbon source or a mixture of D-xylose and D-glucose. S. passalidarum preferentially consumed D-glucose in the co-fermentations, which is consistent with the reduction in expression of genes encoding the key xylose-metabolism enzymes. In the presence of D-glucose, the specific activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) were lower. Interestingly, in accordance with other studies, the presence of 2-deoxyglucose (2DG) did not inhibit the growth of S. passalidarum in culture medium containing D-xylose as the sole carbon source. This indicates that a non-canonical repression pathway is acting in S. passalidarum. In conclusion, the results suggest that D-glucose inhibits D-xylose consumption and prevents the D-xylose-mediated induction of the genes encoding XR, XDH, and XK.

Solar photon-Fenton process eliminates free plasmid DNA harboring antimicrobial resistance genes from wastewater.

This work aimed to assess the elimination and inactivation of resistance-conferring plasmids (RCPs) present in suspension in secondary wastewater by solar photo-Fenton as these are important vectors for the dissemination of antimicrobial resistance. Experiments were performed in synthetic secondary wastewater (SWW) and municipal wastewater treatment plant effluent (MWWTPE). Solar photo-Fenton (50 mg L-1 of H2O2 and 30 mg L-1 of Fe2+) was carried out for 60 min at neutral pH by applying the intermittent iron addition strategy. The removal of RCPs was assessed by Real-Time Polymerase Chain Reaction (qPCR). The transformation of competent non-resistant E. coli was used to evaluate the inactivation of target RCPs harboring antibiotic resistance genes (ARGs) to ampicillin (pSB1A2) or kanamycin (pSB1K3) after treatment and controls. Solar photo-Fenton completely removed RCPs initially present in both matrixes (SWW and MWWTPE), showing enhanced performance compared to the dark Fenton process. Both RCPs were inactivated after 30 min of solar photo-Fenton treatment, while 60 min were necessary to achieve the same effect for the dark Fenton reaction under similar conditions. These results indicate the potential of solar photo-Fenton to improve wastewater quality and reduce the spread of antimicrobial resistance in the environment by hampering the discharge of cell-free RCPs present in suspension in MWWTP onto environmental waters.

Salmonella enterica Optimizes Metabolism After Addition of Acyl-Homoserine Lactone Under Anaerobic Conditions.

Acyl-homoserine lactones (AHLs) are quorum sensing (QS) signaling molecules that mediate cell-to-cell communication in Gram-negative bacteria. Salmonella does not produce AHL, however, it can recognize AHLs produced by other species through SdiA protein modulating important cellular functions. In this work, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on glucose consumption, metabolic profile, and gene expression of Salmonella throughout the cultivation time in Tryptic Soy Broth (TSB) under anaerobic conditions was evaluated. Analysis of the supernatant culture in high-performance liquid chromatography (HPLC) revealed lower glucose uptake after 4 and 6 h of the addition of C12-HSL. Gas chromatography-mass spectrometry (GC-MS) based analysis of the intracellular metabolites revealed C12-HSL perturbation in the abundance levels of metabolites related to the metabolic pathways of glycerolipids, purines, amino acids, and aminoacyl-tRNA biosynthesis. The real-time quantitative PCR (RT-qPCR) indicated that Salmonella increase expression of genes associated with nucleoside degradation and quantification of metabolites supported the induction of pentose phosphate pathway to ensure growth under lower glucose consumption. The obtained data suggest an important role of C12-HSL in the optimization of metabolism at a situation of high population densities.

Identification of B-Cell Epitopes with Potential to Serologicaly Discrimnate Dengue from Zika Infections.

Dengue is currently one of the most important arbovirus infections worldwide. Early diagnosis is important for disease outcome, particularly for those afflicted with the severe forms of infection. The goal of this work was to identify conserved and polymorphic linear B-cell Dengue virus (DENV) epitopes that could be used for diagnostic purposes. To this end, we aligned the predicted viral proteome of the four DENV serotype and performed in silico B-cell epitope mapping. We developed a script in Perl integrating alignment and prediction information to identify potential serotype-specific epitopes. We excluded epitopes that were similarly present in the yellow fever and zika viruses' proteomes. A total of 15 polymorphic and nine conserved peptides among DENV serotypes were selected. Peptides were spotted on cellulose membranes and tested against sera from rabbits that were monoinfected with each DENV serotype. Although serotype-specific peptides failed to recognize any sera, three conserved peptides were recognized by all anti-dengue sera and were included on an ELISA test employing a well-characterized human sera bank. Of the three peptides, one was able to efficiently identify sera from all four DENV serotypes and to discriminate them from Zika virus positive sera.

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-γ and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-γ, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-γ production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease.

Human Exoproteome in Acute Apical Abscesses.

INTRODUCTION: An acute apical abscess is a severe response of the host to massive invasion of the periapical tissues by bacteria from infected root canals. Although many studies have investigated the microbiota involved in the process, information on the host factors released during abscess formation is scarce. The purpose of this study was to describe the human exoproteome in samples from acute apical abscesses. METHODS: Fourteen pus samples were obtained by aspiration from patients with an acute apical abscess. Samples were subjected to protein digestion, and the tryptic peptides were analyzed using a mass spectrometer and ion trap instrument. The human proteins identified in this analysis were classified into different functional categories. RESULTS: A total of 303 proteins were identified. Most of these proteins were involved in cellular and metabolic processes. Immune system proteins were also very frequent and included immunoglobulins, S100 proteins, complement proteins, and heat shock proteins. Polymorphonuclear neutrophil proteins were also commonly detected, including myeloperoxidases, defensins, elastases, and gelatinases. Iron-sequestering proteins including transferrin and lactoferrin/lactotransferrin were found in many samples. CONCLUSIONS: The human exoproteome included a wide variety of proteins related to cellular processes, metabolism, and immune response. Proteins involved in different mechanisms against infection, tissue damage, and protection against tissue damage were identified. Knowledge of the presence and function of these proteins using proteomics provides an insight into the complex host-pathogen relationship, the host antimicrobial strategies to fight infections, and the disease pathogenesis.

A recombinant fusion protein displaying murine and human MHC class I- and II-specific epitopes protects against Leishmania amazonensis infection.

Tegumentary leishmaniasis (TL) constitutes a major public health problem with significant morbidity worldwide. Synthetic peptide-based vaccines are attractive candidates to protect against leishmaniasis, since T cell-specific epitopes can be delivery to antigen-presenting cells, leading to the generation of a Th1 cell-mediated immunity. In this context, the present study aims to evaluate the immunogenicity and protective efficacy of a vaccine composed of major histocompatibility complex class I and II-restricted epitopes derived from four Leishmania infantum proteins to protect mice against Leishmania amazonensis infection. This recombinant fusion protein was administered in BALB/c mice alone or with saponin. As controls, animals received saline or saponin. In the results, the administration of the recombinant protein plus saponin induced a specific IFN-γ, IL-12 and GM-CSF production, as well as high IgG2a isotype antibody levels, which protected mice against a challenge using L. amazonensis promastigotes. Lower parasite burden was found in the infected footpads, liver, spleen and draining lymph node of vaccinated mice, when compared to those from the control groups. In addition, protection was associated with a lower IL-4 and IL-10 response, which was accompanied by the antileishmanial nitrite production by spleen cells of the animals. Interestingly, the recombinant protein administered alone induced a partial protection against challenge. In conclusion, this study shows a new vaccine candidate based on T cell-specific epitopes that was able to induce protection against L. amazonensis infection.

Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI-TcVI. CL Brener, the reference strain of the T. cruzi genome project, is a hybrid with a genome assembled into 41 putative chromosomes. Gene copy number variation (CNV) is well documented as an important mechanism to enhance gene expression and variability in T. cruzi. Chromosomal CNV (CCNV) is another level of gene CNV in which whole blocks of genes are expanded simultaneously. Although the T. cruzi karyotype is not well defined, several studies have demonstrated a significant variation in the size and content of chromosomes between different T. cruzi strains. Despite these studies, the extent of diversity in CCNV among T. cruzi strains based on a read depth coverage analysis has not been determined. RESULTS: We identify the CCNV in T. cruzi strains from the TcI, TcII and TcIII DTUs, by analyzing the depth coverage of short reads from these strains using the 41 CL Brener chromosomes as reference. This study led to the identification of a broader extent of CCNV in T. cruzi than was previously speculated. The TcI DTU strains have very few aneuploidies, while the strains from TcII and TcIII DTUs present a high degree of chromosomal expansions. Chromosome 31, which is the only chromosome that is supernumerary in all six T. cruzi samples evaluated in this study, is enriched with genes related to glycosylation pathways, highlighting the importance of glycosylation to parasite survival. CONCLUSIONS: Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression, which represents a strategy that may be crucial for parasites that mainly depend on post-transcriptional mechanisms to control gene expression.

CD4+ T cells apoptosis in Plasmodium vivax infection is mediated by activation of both intrinsic and extrinsic pathways.

BACKGROUND: Reduction in the number of circulating blood lymphocytes (lymphocytopaenia) has been reported during clinical episodes of malaria and is normalized after treatment with anti-malaria drugs. While this phenomenon is well established in malaria infection, the underlying mechanisms are still not fully elucidated. In the present study, the occurrence of apoptosis and its pathways in CD4+ T cells was investigated in naturally Plasmodium vivax-infected individuals from a Brazilian endemic area (Porto Velho - RO). METHODS: Blood samples were collected from P. vivax-infected individuals and healthy donors. The apoptosis was characterized by cell staining with Annexin V/FITC and propidium iodide and the apoptosis-associated gene expression profile was carried out using RT2 Profiler PCR Array-Human Apoptosis. The plasma TNF level was determined by ELISA. The unpaired t-test or Mann-Whitney test was applied according to the data distribution. RESULTS: Plasmodium vivax-infected individuals present low number of leukocytes and lymphocytes with a higher percentage of CD4+ T cells in early and/or late apoptosis. Increased gene expression was observed for TNFRSF1B and Bid, associated with a reduction of Bcl-2, in individuals with P. vivax malaria. Furthermore, these individuals showed increased plasma levels of TNF compared to malaria-naive donors. CONCLUSIONS: The results of the present study suggest that P. vivax infection induces apoptosis of CD4+ T cells mediated by two types of signaling: by activation of the TNFR1 death receptor (extrinsic pathway), which is amplified by Bid, and by decreased expression of the anti-apoptotic protein Bcl-2 (intrinsic pathway). The T lymphocytes apoptosis could reflect a strategy of immune evasion triggered by the parasite, enabling their persistence but also limiting the occurrence of immunopathology.

Unveiling the intracellular survival gene kit of trypanosomatid parasites.

Trypanosomatids are unicellular protozoans of medical and economical relevance since they are the etiologic agents of infectious diseases in humans as well as livestock. Whereas Trypanosoma cruzi and different species of Leishmania are obligate intracellular parasites, Trypanosoma brucei and other trypanosomatids develop extracellularly throughout their entire life cycle. After their genomes have been sequenced, various comparative genomic studies aimed at identifying sequences involved with host cell invasion and intracellular survival have been described. However, for only a handful of genes, most of them present exclusively in the T. cruzi or Leishmania genomes, has there been any experimental evidence associating them with intracellular parasitism. With the increasing number of published complete genome sequences of members of the trypanosomatid family, including not only different Trypanosoma and Leishmania strains and subspecies but also trypanosomatids that do not infect humans or other mammals, we may now be able to contemplate a slightly better picture regarding the specific set of parasite factors that defines each organism's mode of living and the associated disease phenotypes. Here, we review the studies concerning T. cruzi and Leishmania genes that have been implicated with cell invasion and intracellular parasitism and also summarize the wealth of new information regarding the mode of living of intracellular parasites that is resulting from comparative genome studies that are based on increasingly larger trypanosomatid genome datasets.

Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity.

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a highly debilitating human pathology that affects millions of people in the Americas. The sequencing of this parasite's genome reveals that trans-sialidase/trans-sialidase-like (TcS), a polymorphic protein family known to be involved in several aspects of T. cruzi biology, is the largest T. cruzi gene family, encoding more than 1,400 genes. Despite the fact that four TcS groups are well characterized and only one of the groups contains active trans-sialidases, all members of the family are annotated in the T. cruzi genome database as trans-sialidase. After performing sequence clustering analysis with all TcS complete genes, we identified four additional groups, demonstrating that the TcS family is even more heterogeneous than previously thought. Interestingly, members of distinct TcS groups show distinctive patterns of chromosome localization. Members of the TcSgroupII, which harbor proteins involved in host cell attachment/invasion, are preferentially located in subtelomeric regions, whereas members of the largest and new TcSgroupV have internal chromosomal locations. Real-time RT-PCR confirms the expression of genes derived from new groups and shows that the pattern of expression is not similar within and between groups. We also performed B-cell epitope prediction on the family and constructed a TcS specific peptide array, which was screened with sera from T. cruzi-infected mice. We demonstrated that all seven groups represented in the array are antigenic. A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection. Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.