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The human Respiratory Syncytial Virus (hRSV) stands as one of the most common causes of acute respiratory diseases. The infectivity of this virus is intricately linked to its membrane proteins, notably the attachment glycoprotein (G protein). The latter plays a key role in facilitating the attachment of hRSV to respiratory tract epithelial cells, thereby initiating the infection process. The present study aimed to characterize the interaction of the conserved cysteine-noose domain of hRSV G protein (cndG) with the transmembrane CX3C motif chemokine receptor 1 (CX3CR1) isoforms using computational tools of molecular modeling, docking, molecular dynamics simulations, and binding free energy calculations. From MD simulations of the molecular system embedded in the POPC lipid bilayer, we showed a stable interaction of cndG with the canonical fractalkine binding site in the N-terminal cavity of the CX3CR1 isoforms and identified that residues in the extracellular loop 2 (ECL2) region and Glu279 of this receptor are pivotal for the stabilization of CX3CR1/cndG binding, corroborating what was reported for the interaction of the chemokine fractalkine with CX3CR1 and its structure homolog US28. Therefore, the results presented here contribute by revealing key structural points for the CX3CR1/G interaction, allowing us to better understand the biology of hRSV from its attachment process and to develop new strategies to combat it.

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Protein-protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein-protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide-protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.

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BACKGROUND: The presence of chronic conditions such as type 2 diabetes mellitus (T2DM) requires behavioral lifestyle changes mediated by individuals' motivation for change and adherence to treatment. This study aims to explore activation levels in individuals with T2DM treated in primary care facilities and to identify the association between demographic, clinical, psychosocial factors, and patient activation amongst populations in the Brazilian state of Amazonas. METHODS: SAPPA is a cross-sectional study conducted in Amazonas, approved by the Universidade Federal do Amazona's IRB in Brazil. Individuals with T2DM were evaluated in their homes (n = 4,318,325). The variables were sex, age, skin color, education level; health-related variables such as body mass index, nutritional behavior, and frequency of physical activity. Measures related to patient self-management behaviors over the past 6 months (Patient Activation Measure - PAM-13) were included in the survey. Descriptive and frequency data are presented as mean (standard deviation (SD)) or numeric percentage). Statistical testing was performed using IBM SPSS V.26, and a p-value of < 0.050 showed significance. Activation levels were dichotomized into low activation (Levels 1 and 2) and high activation (Levels 3 and 4). A multivariate linear model assessed the association between the PAM-13 score and the following variables: age, sex, BMI, skin color, number of comorbidities, burden of symptoms, and number of medications. RESULTS: Logistic regression analyses indicated a statistically significant association between sex, age, education, self-rated health, and general satisfaction with life. men were 43% more likely to score lower levels (p < 0.001). The results also indicated that advanced age had lower PAM levels (p < 0.001). Participants with fewer years of education were 44% more likely to have lower levels of PAM (p = 0.03). Worse self-rated health (p < 0.001) and lower general life satisfaction (p = 0.014) were associated with lower PAM levels. CONCLUSIONS: Low patient activation was associated with worse sociodemographic, health, and psychological conditions in the Amazon population. The low level of patient activation observed in this sample highlights an important impediment to diabetes disease management/self-management in disadvantaged populations.

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Protein interactions are at the essence of life. Proteins evolved not to have stable structures, but rather to be specialized in participating in a network of interactions. Every interaction involving proteins comprises the formation of an encounter complex, which may have two outcomes: (i) the dissociation or (ii) the formation of the final specific complex. Here, we present a methodology to characterize the encounter complex of the Grb2-SH2 domain with a phosphopeptide. This method can be generalized to other protein partners. It consists of the measurement of 15N CPMG relaxation dispersion (RD) profiles of the protein in the free state, which describes the residues that are in conformational exchange. We then acquire the dispersion profiles of the protein at a semisaturated concentration of the ligand. At this condition, the chemical exchange between the free and bound state leads to the observation of dispersion profiles in residues that are not in conformational exchange in the free state. This is due to fuzzy interactions that are typical of the encounter complexes. The transient "touching" of the ligand in the protein partner generates these new relaxation dispersion profiles. For the Grb2-SH2 domain, we observed a wider surface at SH2 for the encounter complex than the phosphopeptide (pY) binding site, which might explain the molecular recognition of remote phosphotyrosine. The Grb2-SH2-pY encounter complex is dominated by electrostatic interactions, which contribute to the fuzziness of the complex, but also have contribution of hydrophobic interactions.

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Molecular chaperones aid proteins to fold and assemble without modifying their final structure, requiring, in several folding processes, the interplay between members of the Hsp70 and Hsp40 families. Here, we report the NMR chemical shift assignments for 1 H, 15 N, and 13 C nuclei of the backbone and side chains of the J-domain of the class B Hsp40 from Saccharomyces cerevisiae, Sis1, complexed with the C-terminal EEVD motif of Hsp70. The data revealed information on the structure and backbone dynamics that add significantly to the understanding of the J-domain-Hsp70-EEVD mechanism of interaction.

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AtGRP2 (Arabidopsis thaliana glycine-rich protein 2) is a 19-kDa RNA-binding glycine-rich protein that regulates key processes in A. thaliana. AtGRP2 is a nucleo-cytoplasmic protein with preferential expression in developing tissues, such as meristems, carpels, anthers, and embryos. AtGRP2 knockdown leads to an early flowering phenotype. In addition, AtGRP2-silenced plants exhibit a reduced number of stamens and abnormal development of embryos and seeds, suggesting its involvement in plant development. AtGRP2 expression is highly induced by cold and abiotic stresses, such as high salinity. Moreover, AtGRP2 promotes double-stranded DNA/RNA denaturation, indicating its role as an RNA chaperone during cold acclimation. AtGRP2 is composed of an N-terminal cold shock domain (CSD) followed by a C-terminal flexible region containing two CCHC-type zinc fingers interspersed with glycine-rich sequences. Despite its functional relevance in flowering time regulation and cold adaptation, the molecular mechanisms employed by AtGRP2 are largely unknown. To date, there is no structural information regarding AtGRP2 in the literature. Here, we report the 1H, 15N, and 13C backbone and side chain resonance assignments, as well as the chemical shift-derived secondary structure propensities, of the N-terminal cold shock domain of AtGRP2, encompassing residues 1-90. These data provide a framework for AtGRP2-CSD three-dimensional structure, dynamics, and RNA binding specificity investigation, which will shed light on its mechanism of action.

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Nucleocapsid (NC) assembly is an essential step of the virus replication cycle. It ensures genome protection and transmission among hosts. Flaviviruses are human viruses for which envelope structure is well known, whereas no information on NC organization is available. Here we designed a dengue virus capsid protein (DENVC) mutant in which a highly positive spot conferred by arginine 85 in α4-helix was replaced by a cysteine residue, simultaneously removing the positive charge and restricting the intermolecular motion through the formation of a disulfide cross-link. We showed that the mutant self-assembles into capsid-like particles (CLP) in solution without nucleic acids. Using biophysical techniques, we investigated capsid assembly thermodynamics, showing that an efficient assembly is related to an increased DENVC stability due to α4/α4' motion restriction. To our knowledge, this is the first time that flaviviruses' empty capsid assembly is obtained in solution, revealing the R85C mutant as a powerful tool to understand the NC assembly mechanism.

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Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1H, 15N and 13C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins.

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In this study, we aimed to analyze the effect of 5-fluorouracil, triamcinolone, and bevacizumab on scar modulation in an experimental rat model of surgical lesions. Rats (Rattus norvegicus albinus) were divided into four groups: bevacizumab, 5-fluorouracil + triamcinolone, bevacizumab + 5-fluorouracil + triamcinolone, and control (received no medication) groups. A linear, dorsal incision was created and sutured for the first intention wound healing, mimicking the surgical incision of upper blepharoplasty. Treatments were initiated on day 7, and the rats were euthanized on day 14. Only in the 5-fluorouracil + triamcinolone group was there a difference in the number of infiltrated monocytes. There was 56%, 86%, and 85% decrease in the number of neovessels in the bevacizumab, 5-fluorouracil + triamcinolone, and bevacizumab + 5-fluorouracil + triamcinolone groups, respectively, compared with the control. Picrosirius red staining showed higher collagen density and more organized collagen in the treatment groups than in the control group. Scar modulation was observed in all groups, but the 5-fluorouracil + triamcinolone group presented the best results. To our knowledge, this is the first study to evaluate the influence of three medications in combination on healing. When used together, these medications can prevent the development of unsightly scars, and are therefore promising alternatives to corticosteroids.

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Background: A conservative physiotherapy service development addressed to treat urinary incontinence for older women was studied using the RE-AIM (reach, effectiveness, adoption, implementation, and maintenance) framework. Design: We conducted a pragmatic case study design based on RE-AIM. Settings/participants: Included women ≥ 60 years of age, with self-reported UI symptoms. Results: A total of 34 older women were enrolled in the service with a mean age of 61.53 years. There was a significant improvement in the strength of the pelvic floor muscles, power, endurance, and fast contraction capacity after the intervention, however, it was observed a high dropout rate. Program implementation was supported by Physical Therapy teams who engaged in care coordination. The program has been maintained for over 4 years. Conclusion: Our findings demonstrate that UI patients would benefit from physiotherapy treatment and that this intervention is feasible. This RE-AIM evaluation provides lessons learned and strategies for future adoption, implementation, and maintenance of a Physical Therapy pelvic service.

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Background: Vaccination against COVID-19 was implemented very quickly, but the emergence of new variants that can evade the previous acquired immunological protection highlights the importance of understanding the mechanisms involved in the immune response generated after SARS-CoV-2 infection or vaccination. Objectives: Since most of our knowledge on the humoral immunity generated against SARS-CoV-2 has been obtained from studies with infected patients before vaccination, our goal here was to evaluate seroconversion and its correlation with the titers of neutralizing antibodies (NAbs) in individuals who received the complete initial recommended vaccination schedule with three different vaccines. Study design: We analyzed serum IgG, IgA and total NAbs against the trimeric SARS-CoV-2 Spike (S) protein or its receptor binding domain (RBD) in blood samples collected from 118 healthy individuals without known previous infection, before and after receiving the first and the second dose of CoronaVac (n = 18), ChAdOx-1 (n = 68) or BNT162b2 (n = 32) vaccines. Results: We found that although IgG titers were high in all sera collected after the two doses of these vaccines, NAbs amounts varies among the groups. In contrast, serum NAbs concentrations were much more comparable to the IgA levels, indicating that these antibodies would have a major neutralizing capacity against SARS-CoV-2. Conclusions: Altogether our data suggest that quantification of serum anti-S or anti-RBD IgA, rather than IgG, may be a valuable tool to screen NAbs and may be considered for surveillance of vaccine coverage.

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Introduction: The effective translation of evidence-based interventions has contributed to implementing actions that impact public policies and the population's health. However, there is a gap in the literature regarding the factors associated with the successful implementation of these interventions. The Active Life Improving Health Behavior Change Program (BCP-VAMOS) uses behavioral strategies to promote an active and healthy lifestyle in the community. Characterized as a health innovation, it also provides health professionals with online training to implement the program in Primary Health Care (PHC). Our study describes a pragmatic trial that aims to evaluate the implementation of BCP-VAMOS, version 3.0, in PHC in southern Brazil. Methods and analysis: A pragmatic randomized controlled trial (PRCT) of two arms comparing a group of PHC professionals who will participate in a traditional didactic approach (control group) vs. a group that will receive ongoing support (intervention group) for the implementation of BCP-VAMOS. The intervention will be available to adults (≥18 years old) registered at PHC. Program recipient's will be assessed at baseline and post-intervention (9 months after) to measure markers of physical activity and eating behavior (primary outcomes). Program's implementation process will be monitored for 12 months and will be evaluated using the RE-AIM and Consolidated Framework for Implementation Research (CFIR) frameworks. Discussions: The survey findings can be used widely throughout Brazil, guiding the work of health professionals, service planners and policy-makers. Also, the results may help to inform the national health promotion policy to plan interventions and improve the implementation of programs in PHC. This research results will provide practical guidance for researchers to develop similar protocols to implement and adapt public health interventions. Ethics and dissemination: Ethics approval has been granted by the Human Research Ethics Committee of the Federal University of Santa Catarina (UFSC), Brazil, under no. 1394492. Results will be published in full as open access in the UFSC library and main trial results and associated papers in high-impact peer-reviewed journals. Trial registration number: RBR-2vw77q-Brazilian Registry of Clinical Trials - ReBEC (http://www.ensaiosclinicos.gov.br).

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BACKGROUND: Changes in the profiles of patients have significant impacts on the health care system. Diabetes mellitus type 2 (T2DM) prevention and management should be studied in different contexts. OBJECTIVE: The Study of Health in Primary Care for the Amazonas Population (SAPPA) primarily aims to describe T2DM prevention and management actions offered by primary health care settings in Brazil and whether the care delivered is consistent with the chronic care model (CCM). Second, the study aims to examine the impact of T2DM management actions on health and lifestyle, and third, to understand how sociodemographic characteristics, health, and subjective outcomes impact diabetes management. METHODS: As part of this observational study, managers and health professionals complete a questionnaire containing information about T2DM prevention and management actions and CCM dimensions. During in-home visits, patients are asked about their health, lifestyle, sociodemographics, diabetes care, and subjective variables. RESULTS: A total of 34 managers, 1560 professional health workers, and 955 patients will be recruited. The data collection will be completed in October 2022. CONCLUSIONS: The SAPPA is an observational study that intends to understand the T2DM management process in primary health care, including planning, execution, reach, and impact on patient motivation and adherence. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/37572.

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Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M ß-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried ß-sheet. The two mutations located in this ß-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M ß-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.

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Nitric oxide (NO) is a free radical with a signaling capacity. Its cellular functions are achieved mainly through S-nitrosation where thioredoxin (hTrx) is pivotal in the S-transnitrosation to specific cellular targets. In this study, we use NMR spectroscopy and mass spectrometry to follow the mechanism of S-(trans)nitrosation of hTrx. We describe a site-specific path for S-nitrosation by measuring the reactivity of each of the 5 cysteines of hTrx using cysteine mutants. We showed the interdependence of the three cysteines in the nitrosative site. C73 is the most reactive and is responsible for all S-transnitrosation to other cellular targets. We observed NO internal transfers leading to C62 S-nitrosation, which serves as a storage site for NO. C69-SNO only forms under nitrosative stress, leading to hTrx nuclear translocation.

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Introduction: The relationship between obesity and physical activity is still unknown in specific groups, such as people living with ostomies. Objective: To assess the prevalence of obesity and the level of physical activity in ostomized individuals. Methods: A cross-sectional study with a population of ostomized patients in two hospitals of a Brazilian city. Obesity was assessed by the Body Mass Index (BMI) and physical activity, by the International Physical Activity Questionnaire. Results: The study included 148 patients (55.4% of men [82]), with a mean age of 58.5 (±17.8) years, with 56.1% (83) of the sample aged 60 or older. Colostomies were identified as definitive in 67.6% (100), there were 28 cases (18.9%) of obesity, 67 (45.3%) overweight patients, and 31 (20.9%) subjects were underweight. Sedentary lifestyle was identified in 40.5% (60) of the participants. The mean BMI was significantly higher in men (25.9 ± 5.1), and a higher proportion of men were overweight (28; 34.1%); the underweight group had a greater proportion of women (20; 30.3%). An association between physical inactivity and underweight (p = 0.003) was found. Conclusion: Most patients with ostomies, especially colostomies, are men over 60 years of age who are overweight or obese. In general, underweight individuals are more sedentary. (AU)

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Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an acute febrile and arthritogenic illness with no effective treatments available. The development of effective therapeutic strategies could be significantly accelerated with detailed knowledge of the molecular components behind CHIKV replication. However, drug discovery is hindered by our incomplete understanding of their main components. The RNA-dependent RNA-polymerase (nsP4-CHIKV) is considered the key enzyme of the CHIKV replication complex and a suitable target for antiviral therapy. Herein, the nsP4-CHIKV was extensively characterized through experimental and computational biophysical methods. In the search for new molecules against CHIKV, a compound designated LabMol-309 was identified as a strong ligand of the nsp4-CHIKV and mapped to bind to its active site. The antiviral activity of LabMol-309 was evaluated in cellular-based assays using a CHIKV replicon system and a reporter virus. In conclusion, this study highlights the biophysical features of nsP4-CHIKV and identifies a new compound as a promising antiviral agent against CHIKV infection.

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The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens.

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Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signaling and metabolism to favor bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes three VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. A total of 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin and induces actin remodeling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis.

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