Characterization of an antagonistic peptide produced by a Bacteroides Fragilis isolate obtained from a patient with intra-abdominal infection / Caracterização de um peptido antagonístico produzido por Bacteroides Fragilis isolado obtido em um paciente com infecção intra-abdominal

The indigenous microbiota of humans is extremely rich and diverse, with special emphasis on the intestinal microbiota. As a constituent of this microbiota, it is mentioned the genus Bacteroides, whose members are Gram negative rods, obligate anaerobes, amphibionts, associated to the etiopathogenesis of important infectious diseases, such as intra-abdominal infections. Bacteroides strains have the ability to synthesize antagonistic substances that play an ecological role, especially in densely colonized habitats, giving a competitive advantage to the producing samples. The objective of this study was to evaluate the synthesis capacity of antagonistic substances by 40 samples of Bacteroides and Parabacteroides isolated from patients with intra-abdominal infections. The expression of antagonism was evaluated by the overlay diffusion method, using, as well as the test samples, 36 reference samples of Gram negative and Gram-positive bacteria. Subsequently, a production strain (Bacteroides fragilis) was used for extraction, purification and partial characterization of the detected antagonistic substance. As indicator strains, Bacteroides ovatus and Bacteroides caccae were used. The production strain was submitted to protein extraction, and the activity of the precipitated intracellular extract was detected with (NH4) 2SO4 in concentrations of 30% (C30) and 50% (C50). C30 and C50 were inactivated by proteases and high temperatures and remained active after exposure to organic solvents and a wide pH range. Both fractions presented antagonistic activity of bacteriostatic nature. The C50 extract was subjected to ion exchange chromatography, and 50 fractions were recovered. Among them, fractions 1 to 4, referring to a single peak, that were not able to bind to the column, presented antagonistic activity. Fractions from the ion exchange chromatography were applied in gel filtration chromatography. Among them, fractions 2 and 3 were able to inhibit the developing sample. These fractions were submitted to reverse phase chromatography, and 50 fractions were collected. One of them, fraction 2C, remained active against the revealing sample. Mass spectrometry, from fraction 2C obtained from reverse phase chromatography, presented ions of approximately 1300.00 Da, which generated a more intense signal. The search performed by similarity between the sequenced peptides and proteins described in the BLASTP database, from fragmentation obtained in reverse phase chromatography, resulted in 100% identity between two peptides. One of the sequenced peptides showed 100% identity to a type VII secretion protein. The search performed by similarity between the sequenced peptides and proteins described in the ANTIMICROBIAL PEPTIDE DATABASE database, from fragmentation obtained with trypsin digestion, resulted in 42% identity with a Streptomyces microcine. Together, the results indicate the production of antagonistic substances by the B. fragilis sample under study. It is plausible to assume that they play a relevant role in interbacterial relationships, as a virulence factor, in a complex environment such as intra-abdominal infection

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-? (IFN-?), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-? (TNF-?), 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-? 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-?, 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-? 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

Burden of disease scenarios for 204 countries and territories, 2022–2050: a forecasting analysis for the Global Burden of Disease Study 2021

Background: Future trends in disease burden and drivers of health are of great interest to policy makers and the public at large. This information can be used for policy and long-term health investment, planning, and prioritisation. We have expanded and improved upon previous forecasts produced as part of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) and provide a reference forecast (the most likely future), and alternative scenarios assessing disease burden trajectories if selected sets of risk factors were eliminated from current levels by 2050. Methods: Using forecasts of major drivers of health such as the Socio-demographic Index (SDI; a composite measure of lag-distributed income per capita, mean years of education, and total fertility under 25 years of age) and the full set of risk factor exposures captured by GBD, we provide cause-specific forecasts of mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) by age and sex from 2022 to 2050 for 204 countries and territories, 21 GBD regions, seven super-regions, and the world. All analyses were done at the cause-specific level so that only risk factors deemed causal by the GBD comparative risk assessment influenced future trajectories of mortality for each disease. Cause-specific mortality was modelled using mixed-effects models with SDI and time as the main covariates, and the combined impact of causal risk factors as an offset in the model. At the all-cause mortality level, we captured unexplained variation by modelling residuals with an autoregressive integrated moving average model with drift attenuation. These all-cause forecasts constrained the cause-specific forecasts at successively deeper levels of the GBD cause hierarchy using cascading mortality models, thus ensuring a robust estimate of cause-specific mortality. For non-fatal measures (eg, low back pain), incidence and prevalence were forecasted from mixed-effects models with SDI as the main covariate, and YLDs were computed from the resulting prevalence forecasts and average disability weights from GBD. Alternative future scenarios were constructed by replacing appropriate reference trajectories for risk factors with hypothetical trajectories of gradual elimination of risk factor exposure from current levels to 2050. The scenarios were constructed from various sets of risk factors: environmental risks (Safer Environment scenario), risks associated with communicable, maternal, neonatal, and nutritional diseases (CMNNs; Improved Childhood Nutrition and Vaccination scenario), risks associated with major non-communicable diseases (NCDs; Improved Behavioural and Metabolic Risks scenario), and the combined effects of these three scenarios. Using the Shared Socioeconomic Pathways climate scenarios SSP2-4.5 as reference and SSP1-1.9 as an optimistic alternative in the Safer Environment scenario, we accounted for climate change impact on health by using the most recent Intergovernmental Panel on Climate Change temperature forecasts and published trajectories of ambient air pollution for the same two scenarios. Life expectancy and healthy life expectancy were computed using standard methods. The forecasting framework includes computing the age-sex-specific future population for each location and separately for each scenario. 95% uncertainty intervals (UIs) for each individual future estimate were derived from the 2·5th and 97·5th percentiles of distributions generated from propagating 500 draws through the multistage computational pipeline. Findings: In the reference scenario forecast, global and super-regional life expectancy increased from 2022 to 2050, but improvement was at a slower pace than in the three decades preceding the COVID-19 pandemic (beginning in 2020). Gains in future life expectancy were forecasted to be greatest in super-regions with comparatively low life expectancies (such as sub-Saharan Africa) compared with super-regions with higher life expectancies (such as the high-income super-region), leading to a trend towards convergence in life expectancy across locations between now and 2050. At the super-region level, forecasted healthy life expectancy patterns were similar to those of life expectancies. Forecasts for the reference scenario found that health will improve in the coming decades, with all-cause age-standardised DALY rates decreasing in every GBD super-region. The total DALY burden measured in counts, however, will increase in every super-region, largely a function of population ageing and growth. We also forecasted that both DALY counts and age-standardised DALY rates will continue to shift from CMNNs to NCDs, with the most pronounced shifts occurring in sub-Saharan Africa (60·1% [95% UI 56·8–63·1] of DALYs were from CMNNs in 2022 compared with 35·8% [31·0–45·0] in 2050) and south Asia (31·7% [29·2–34·1] to 15·5% [13·7–17·5]). This shift is reflected in the leading global causes of DALYs, with the top four causes in 2050 being ischaemic heart disease, stroke, diabetes, and chronic obstructive pulmonary disease, compared with 2022, with ischaemic heart disease, neonatal disorders, stroke, and lower respiratory infections at the top. The global proportion of DALYs due to YLDs likewise increased from 33·8% (27·4–40·3) to 41·1% (33·9–48·1) from 2022 to 2050, demonstrating an important shift in overall disease burden towards morbidity and away from premature death. The largest shift of this kind was forecasted for sub-Saharan Africa, from 20·1% (15·6–25·3) of DALYs due to YLDs in 2022 to 35·6% (26·5–43·0) in 2050. In the assessment of alternative future scenarios, the combined effects of the scenarios (Safer Environment, Improved Childhood Nutrition and Vaccination, and Improved Behavioural and Metabolic Risks scenarios) demonstrated an important decrease in the global burden of DALYs in 2050 of 15·4% (13·5–17·5) compared with the reference scenario, with decreases across super-regions ranging from 10·4% (9·7–11·3) in the high-income super-region to 23·9% (20·7–27·3) in north Africa and the Middle East. The Safer Environment scenario had its largest decrease in sub-Saharan Africa (5·2% [3·5–6·8]), the Improved Behavioural and Metabolic Risks scenario in north Africa and the Middle East (23·2% [20·2–26·5]), and the Improved Nutrition and Vaccination scenario in sub-Saharan Africa (2·0% [–0·6 to 3·6]). Interpretation: Globally, life expectancy and age-standardised disease burden were forecasted to improve between 2022 and 2050, with the majority of the burden continuing to shift from CMNNs to NCDs. That said, continued progress on reducing the CMNN disease burden will be dependent on maintaining investment in and policy emphasis on CMNN disease prevention and treatment. Mostly due to growth and ageing of populations, the number of deaths and DALYs due to all causes combined will generally increase. By constructing alternative future scenarios wherein certain risk exposures are eliminated by 2050, we have shown that opportunities exist to substantially improve health outcomes in the future through concerted efforts to prevent exposure to well established risk factors and to expand access to key health interventions

Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) - doi: 10.4025/actascibiolsci.v34i3.9239

Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors

Association of casein micelle size and enzymatic curd strength and dry matter curd yield

ABSTRACT: The aim of the present study was to explore the association between milk protein content and casein micelle size and to examine the effects of casein micelle size on enzymatic curd strength and dry matter curd yield using reduced laboratory-scale cheese production. In this research, 140 bulk tank milk samples were collected at dairy farms. The traits were analyzed using two linear models, including only fixed effects. Smaller micelles were associated with higher κ-casein and lower αs-casein contents. The casein micellar size (in the absence of the αs-casein and κ-casein effects) did not affect the enzymatic curd strength; however, smaller casein micelles combined with higher fat, lactose, casein and κ-casein contents exhibited a favorable effect on the dry matter curd yield. Overall, results of the present study provide new insights into the importance of casein micelle size for optimizing cheese production

A novel and efficient and low-cost methodology for purification of Macrotyloma axillare (Leguminosae) seed lectin.

The N-acetyl-galactosamine specific lectin fromMacrotyloma axillare seeds (LMA)was purified by precipitation and ion exchange chromatography. The LMA 0.2 mol L−1 fraction showed hemagglutinating activity on erythrocytes A1. The results for molecular mass determinations were about 28 kDa. The LMA pHdependent assays showed best hemagglutinating activity at pH 6.0–8.0; being decreased at acidic/alkaline conditions and by EDTA treatment. LMA is a tetramer at pH 8.2 and a dimer at pH 4.0. Human erythrocytes from ABO system confirmed the A1 specificity for LMA. This new methodology is useful and easy, with low costs, for lectin purification in large amounts

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease

Immunodiagnosis of human and canine visceral leishmaniasis using recombinant Leishmania infantum Prohibitin protein and a synthetic peptide containing its conformational B-cell epitope.

In the present study, Leishmania infantum's Prohibitin was cloned and, alongside a synthetic peptide, evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis (CVL and TL, respectively) in dogs and humans. For TL diagnosis, this study analyzed serum samples from cutaneous (n=20) or mucosal (n=39) leishmaniasis patients, and from Chagas disease (CD) patients (n=8) and non-infected patients (n=45). For CVL diagnosis, serum samples from asymptomatic (n=14), symptomatic (n=71), non-infected (n=116), and Leish-Tec?- vaccinated (n=79) dogs were examined, as well as T. cruzi (n=11) and Ehrlichia canis (n=10) infected animals. An indirect ELISA method using rProhibitin showed diagnostic sensitivity and specificity values of 91.76% and 89.91%, respectively. L. infantum SLA showed 86.11% and 48.24% of specificity and sensitivity, respectively, for CVL serodiagnosis, and 98.31% and 84.91% sensitivity and specificity, respectively for TL diagnosis. L. braziliensis SLA showed 75.47% and 83.05% of specificity and sensitivity, respectively, for TL diagnosis. The synthetic peptide showed a better result in TL than in CVL diagnosis. In conclusion, preliminar results suggest that the detection of antibodies against the rProhibitin protein and the synthetic peptide improves the serodiagnosis of TL and CVL

A Leishmania hypothetical protein-containing liposome-based formulation is highly immunogenic and induces protection against visceral leishmaniasis.

Leishmania proteins have been evaluated as vaccine candidates against leishmaniasis; however, most antigens present low immunogenicity and need to be added with immune adjuvants. A low number of licensed adjuvants exist on the market today; therefore, research conducted to produce new products is desirable. The present study sought to evaluate the immunogenicity and protective efficacy of a recombinant Leishmania hypothetical protein, namely LiHyR, administered with saponin or liposomes in BALB/c mice. Immunological and parasitological parameters were evaluated, and results showed significant protection against Leishmania infantum infection produced by both compositions in the immunized animals; however, this was not identified when the antigen was used alone. In addition, the liposomal formulation was more effective in inducing a polarized Th1 response in the vaccinated animals, which was maintained after challenge and reflected by lower parasitism found in all evaluated organs when the limiting dilution technique and RT-PCR assay were employed. The protected animals showed higher levels of protein and parasite-specific IFN-? IL-2, IL-12, GM-CSF, and TNF-?, which were evaluated by capture ELISA and flow cytometry, in addition to a higher production of anti-protein and anti-parasite IgG2a antibodies, both before and after challenge. The Lip/rLiHyR combination induced higher IFN-? production through both CD4+ and CD8+ T cell subtypes. Results indicate the possibility of using the LiHyR, containing a liposomal formulation, as a vaccine candidate against visceral leishmaniasis