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Abstract Background Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. Methods To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl −/− ) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). Results We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl −/− mice as compared to wild-type. Rankl −/− mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl −/− SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. Conclusions We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

Resumen: Los cuidados paliativos son «responsabilidad ética de los sistemas de salud». La OMS propone la educación médica de grado y posgrado como una dimensión para evaluar su desarrollo en un país. En Latinoamérica, solo el 15% de las facultades de medicina tienen una asignatura de Cuidados Paliativos. En Argentina no existe aún consenso sobre contenidos básicos en los currículos de grado en Medicina. Método: este estudio buscó consensuar cómo debe ser un currículo de grado en Medicina Paliativa en Argentina utilizando la metodología Delphi. Se recogieron datos sociodemográficos (2019–2020), profesión, años de experiencia, educación universitaria, contenidos temáticos, modalidad pedagógica, plan de estudios y carga horaria. El análisis cuantitativo se realizó utilizando porcentajes y valores medios para indicar el nivel de acuerdo en las escalas de 5 puntos. Los rangos intercuartílicos se calcularon para proporcionar indicadores de acuerdo y consenso. Se realizó el análisis temático de contenidos. Resultados: se convocaron 250 expertos, de los cuales 85 respondieron a 2 rondas de consulta. Se alcanzó un alto grado de acuerdo para proponer 10 recomendaciones generales, dinámicas, flexibles, adaptadas a diferentes contextos. El 98% consideró necesaria la enseñanza de los cuidados paliativos de manera transversal y sugirieron una asignatura específica contextualizada a situaciones de pacientes crónicos y en fin de vida. Sin embargo, se observó una tensión entre el modelo de formación tradicional y la educación basada en competencias. El aprendizaje experiencial es apropiado, pero se recomendó la unión de la teoría con la práctica. Conclusión: por primera vez en Argentina se convocó un consenso de expertos que definió un conjunto de recomendaciones sobre contenidos, metodología de enseñanza, ciclo académico y competencias en cuidados paliativos. Abstract: Palliative care is an “ethical responsibility of healthcare systems”. The WHO proposes undergraduate and postgraduate medical education as one dimension to assess its development in a country. In Latin America, 15% of medical schools have an undergraduate palliative care course. In Argentina, there is still no consensus on the essential content of undergraduate medical curricula. Method: This study sought to reach a consensus on what an undergraduate curriculum in palliative medicine should look like in Argentina using the Delphi methodology (2019–2020). Sociodemographic data, profession, years of experience, university education, thematic content, pedagogical modality, study plan and hourly load were collected. Quantitative analysis was performed using percentages and mean values to indicate the level of agreement on 5-point scales. Interquartile ranges were calculated to provide indicators of agreement and consensus. Thematic content analysis was carried out. Results: Two hundred fifty experts were convened, of whom 85 responded to two rounds of consultation. A high degree of agreement was reached on ten general, dynamic, and flexible recommendations adapted to different contexts. 98% considered it necessary to teach palliative care transversally and suggested a specific course contextualised to chronic patients and end-of-life situations. However, there was a tension between the traditional training model and competency-based education. Experiential learning is appropriate, but linking theory and practice was recommended. Conclusion: For the first time in Argentina, a consensus of experts was reached, which defined a set of recommendations on content, teaching methodology, academic cycle and competencies in palliative care.

Lyme disease, a zoonotic infection caused by the bacterium Borrelia burgdorferi, is transmitted to humans through the bites of infected ticks. Its diagnosis primarily relies on serological methods; however, the existing borreliosis techniques have shown a variable sensitivity and specificity. Our study aimed to map IgG epitopes from five outer membrane proteins (Omp) from B. burgdorferi [Filament flagellar 41kD (PI1089), flagellar hook-associated protein (Q44767), Flagellar hook k2 protein (O51173), Putative Omp BURGA03 (Q44849), and 31 kDa OspA (P0CL66)] lipoprotein to find specific epitopes for the development of accurate diagnosis methods. Using the spot synthesis technique, a library of 380 peptides was constructed to identify linear B cell epitopes recognized by human IgG in response to specific B. burgdorferi-associated proteins. The reactivity of this epitope when chemically synthesized was then evaluated using ELISA with a panel of the patient’s sera. Cross-reactivity was assessed through data bank access and in vitro analysis. Among the 19 epitopes identified, four were selected for further investigation based on their signal intensity, secondary structure, and peptide matching. Validation was performed using ELISA, and ROC curve analysis demonstrated a sensitivity of ≥85.71%, specificity of ≥92.31, accuracy of ≥90.7, and AUC value of ≥0.91 for all peptides. Our cross-reactivity analysis demonstrated that the Burg/02/huG, Burg/03/huG, and Burg/12/huG peptides were not reactive to antibodies from patients with Leptospirosis and syphilis compared to those from the B. burgdorferi group. These peptides indicated an excellent performance in distinguishing between B. burgdorferi-infected and non-infected individuals and exhibited a neglected reactivity to antibodies in sera from patients with Leptospirosis and syphilis. These peptides are promising targets for recombinant development, potentially leading to more accurate serological tests and vaccines.

Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases.

Introdução: Dentre as doenças imunopreveníveis bacterianas estão a difteria, o tétano e a coqueluche, essas doenças mesmo com vacinas disponíveis pelo Programa Nacional de Imunização (PNI) tem sido motivo de preocupação, destaca-se os movimentos antivacinas e queda na cobertura vacinal. No Brasil, 1,6 milhão de crianças não receberam nenhuma dose da vacina DTP, que previne contra difteria, tétano e coqueluche, entre 2019 e 2021. Levando assim a preocupação do aumento de casos dessas doenças, como destaque para a coqueluche, bem com a possibilidade da ressurgência da difteria em nosso país, principalmente devido a baixa cobertura vacinal em algumas regiões e também agravado com a ocorrência de inúmeros casos na América Latina, como observado na Venezuela nos últimos anos. Objetivo: Identificar os principais epítopos imuno dominantes das toxinas Pertussis, diftérica e tetânica contribuindo assim para o desenvolvimento de novos testes diagnósticos e o aperfeiçoamento de novos imunobiológicos. Métodos: Foram realizadas análises de alta resolução dos epítopos lineares de células B das toxinas em estudos. Membrana de celulose através da síntese SPOT e sondados com soros de crianças vacinadas (DTP) foram posteriormente caracterizados e validados usando imunoensaios. Resultados: Um conjunto de 43 epítopos lineares de células B reconhecidos pela IgG humana após a administração de uma vacina DTP em crianças foram identificados para a toxina tetânica, 20 epítopos foram reconhecidos para a toxina diftérica e 24 epítopos foram identificados para toxinas Pertussis. Conclusão: Neste estudo identificamos todos os epítopos B lineares dos imunógenos das toxinas pertussis, diftérica e tetânica. A identificação e o mapeamentos dos epítopos poderão contribuir para o desenvolvimento de métodos de diagnósticos mais eficientes. Resultados desse trabalho também poderão contribuir para entendermos o processo de imunização e o aperfeiçoamento de imunobiológicos para que sejam mais eficientes e menos reatogências.

Introdução: A coqueluche é uma doença respiratória altamente contagiosa causada pela bactéria Bordetella pertussis. A doença é mais comum em crianças, mas pode afetar pessoas de qualquer idade. Segundo a Organização Mundial da Saúde (OMS), a doença é endêmica em todo o mundo, afetando cerca de 24,1 milhões de pessoas e causando cerca de 160.700 mortes por ano. No entanto, estima-se que esses números podem ser maiores, devido à subnotificação dos casos. Objetivo: O estudo teve como objetivo avaliar níveis de IgG circulante em crianças na faixa etária de 1 a 13 anos que foram imunizadas com vacinas do Programa Nacional de Imunização (PNI) no município de Duque de Caxias, RJ. Métodos: Foram analisados 225 soros de crianças na faixa etária de 1 a 13 anos sem evidência de infecção aguda ou história conhecida de tosse convulsa e difteria. Os soros foram analisados através do teste de Elisa para detecção de IgG circulante para toxinas Pertussis e também para componentes da Bordetella pertussis. Este trabalho foi aprovado pelo comitê de ética do centro de estudos UNIGRANRIO (CAAE: 24856610.0.0000.5283) e conduzido de acordo com as boas práticas clínicas e todos os requisitos regulamentares aplicáveis, incluindo a Declaração de Helsinque. Resultados: Os resultados encontrados demostram que a maioria da IgG circulantes nos soros analisados tanto para a toxina Pertussis como para a Bordetella Pertussis em todas as faixas etárias estão abaixo dos níveis esperados para manter uma boa proteção, favorecendo assim uma nova infecção. Conclusão: Destaca se a necessidade de realizar novos estudos com a participação de outros municípios, onde poderemos observar se a realidade encontrada no município de Duque de Caxias reflete a realidade de todo Estado do Rio de Janeiro ou é apenas uma característica local. Assim novas medidas poderão ser adotadas com intuito de aumentar a resposta imunológica da população, principalmente utilizado dose de reforço com vacina DTP com também o aprimoramento da fração Pertussis na composição da vacina utilizado no Programa Nacional de Imunização.

The worldwide spread of SARS-CoV-2 has led to a significant economic and social burden on a global scale. Even though the pandemic has concluded, apprehension remains regarding the emergence of highly transmissible variants capable of evading immunity induced by either vaccination or prior infection. The success of viral penetration is due to the specific amino acid residues of the receptor-binding motif (RBM) involved in viral attachment. This region interacts with the cellular receptor ACE2, triggering a neutralizing antibody (nAb) response. In this study, we evaluated serum immunogenicity from individuals who received either a single dose or a combination of different vaccines against the original SARS-CoV-2 strain and a mutated linear RBM. Despite a modest antibody response to wild-type SARS-CoV-2 RBM, the Omicron variants exhibit four mutations in the RBM (S477N, T478K, E484A, and F486V) that result in even lower antibody titers. The primary immune responses observed were directed toward IgA and IgG. While nAbs typically target the RBD, our investigation has unveiled reduced seroreactivity within the RBD’s crucial subregion, the RBM. This deficiency may have implications for the generation of protective nAbs. An evaluation of S1WT and S2WT RBM peptides binding to nAbs using microscale thermophoresis revealed a higher affinity (35 nM) for the S2WT sequence (GSTPCNGVEGFNCYF), which includes the FNCY patch. Our findings suggest that the linear RBM of SARS-CoV-2 is not an immunodominant region in vaccinated individuals. Comprehending the intricate dynamics of the humoral response, its interplay with viral evolution, and host genetics is crucial for formulating effective vaccination strategies, targeting not only SARS-CoV-2 but also anticipating potential future coronaviruses.

Background: The newly introduced COVID-19 vaccines have reduced disease severity and hospitalizations. However, they do not significantly prevent infection or transmission. In the same context, measuring IgM and IgG antibody levels is important, but it does not provide information about the status of the mucosal immune response. This article describes a comprehensive mapping of IgA epitopes of the S protein, its cross-reactivity, and the development of an ELISA-peptide assay. Methods: IgA epitope mapping was conducted using SPOT synthesis and sera from RT-qPCR COVID-19-positive patients. Specific and cross-reacting epitopes were identified, and an evolutionary analysis from the early Wuhan strain to the Omicron variant was performed using bioinformatics tools and a microarray of peptides. The selected epitopes were chemically synthesized and evaluated using ELISA-IgA. Results: A total of 40 IgA epitopes were identified with 23 in S1 and 17 in the S2 subunit. Among these, at least 23 epitopes showed cross-reactivity with DENV and other organisms and 24 showed cross-reactivity with other associated coronaviruses. Three MAP4 polypeptides were validated by ELISA, demonstrating a sensitivity of 90–99.96% and a specificity of 100%. Among the six IgA-RBD epitopes, only the SC/18 epitope of the Omicron variants (BA.2 and BA.2.12.1) presented a single IgA epitope. Conclusions: This research unveiled the IgA epitome of the S protein and identified many epitopes that exhibit cross-reactivity with DENV and other coronaviruses. The S protein of variants from Wuhan to Omicron retains many conserved IgA epitopes except for one epitope (#SCov/18). The cross-reactivity with DENV suggests limitations in using the whole S protein or the S1/S2/RBD segment for IgA serological diagnostic tests for COVID-19. The expression of these identified specific epitopes as diagnostic biomarkers could facilitate monitoring mucosal immunity to COVID-19, potentially leading to more accurate diagnoses and alternative mucosal vaccines.

The rise in antibiotic-resistant strains of clinically important pathogens is a major threat to global health. The World Health Organization (WHO) has recognized the urgent need to develop alternative treatments to address the growing list of priority pathogens. Antimicrobial peptides (AMPs) rank among the suggested options with proven activity and high potential to be developed into effective drugs. Many AMPs are naturally produced by living organisms protecting the host against pathogens as a part of their innate immunity. Mechanisms associated with AMP actions include cell membrane disruption, cell wall weakening, protein synthesis inhibition, and interference in nucleic acid dynamics, inducing apoptosis and necrosis. Acinetobacter baumannii is a critical pathogen, as severe clinical implications have developed from isolates resistant to current antibiotic treatments and conventional control procedures, such as UV light, disinfectants, and drying. Here, we review the natural AMPs representing primary candidates for new anti-A. baumannii drugs in post-antibiotic-era and present computational tools to develop the next generation of AMPs with greater microbicidal activity and reduced toxicity.

Introduction Adequately addressing the needs of patients at the end of life and their relatives is pivotal in preventing unnecessary suffering and optimising their quality of life. The purpose of the iLIVE study is to contribute to high-quality personalised care at the end of life in different countries and cultures, by investigating the experiences, concerns, preferences and use of care of terminally ill patients and their families.Methods and analysis The iLIVE study is an international cohort study in which patients with an estimated life expectancy of 6 months or less are followed up until they die. In total, 2200 patients will be included in 11 countries, that is, 200 per country. In addition, one relative per patient is invited to participate. All participants will be asked to fill in a questionnaire, at baseline and after 4 weeks. If a patient dies within 6 months of follow-up, the relative will be asked to fill in a post-bereavement questionnaire. Healthcare use in the last week of life will be evaluated as well; healthcare staff who attended the patient will be asked to fill in a brief questionnaire to evaluate the care that was provided. Qualitative interviews will be conducted with patients, relatives and healthcare professionals in all countries to gain more in-depth insights.Ethics and dissemination The cohort study has been approved by ethics committees and the institutional review boards (IRBs) of participating institutes in all countries. Results will be disseminated through the project website, publications in scientific journals and at conferences. Within the project, there will be a working group focusing on enhancing the engagement of the community at large with the reality of death and dying.Trial registration number NCT04271085.

Abstract Mayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

Tetanus is an acute, fatal disease caused by exotoxins released from Clostridium tetani during infections. A protective humoral immune response can be induced by vaccinations with pediatric and booster combinatorial vaccines that contain inactivated tetanus neurotoxin (TeNT) as a major antigen. Although some epitopes in TeNT have been described using various approaches, a comprehensive list of its antigenic determinants that are involved with immunity has not been elucidated. To this end, a high-resolution analysis of the linear B-cell epitopes in TeNT was performed using antibodies generated in vaccinated children. Two hundred sixty-four peptides that cover the entire coding sequence of the TeNT protein were prepared in situ on a cellulose membrane through SPOT synthesis and probed with sera from children vaccinated (ChVS) with a triple DTP-vaccine to map continuous B-cell epitopes, which were further characterized and validated using immunoassays. Forty-four IgG epitopes were identified. Four (TT-215-218) were chemically synthesized as multiple antigen peptides (MAPs) and used in peptide ELISAs to screen post-pandemic DTP vaccinations. The assay displayed a high performance with high sensitivity (99.99%) and specificity (100%). The complete map of linear IgG epitopes induced by vaccination with inactivated TeNT highlights three key epitopes involved in the efficacy of the vaccine. Antibodies against epitope TT-8/G can block enzymatic activity, and those against epitopes TT-41/G and TT-43/G can interfere with TeNT binding to neuronal cell receptors. We further show that four of the epitopes identified can be employed in peptide ELISAs to assess vaccine coverage. Overall, the data suggest a set of select epitopes to engineer new, directed vaccines.

Zoonotic-origin infectious diseases are one of the major concerns of human and veterinary health systems. Ticks, as vectors of several zoonotic diseases, are ranked second only to mosquitoes as vectors. Many ticks’ transmitted infections are still endemic in the Americas, Europe, and Africa and represent approximately 17% of their infectious diseases population. Although our scientific capacity to identify and diagnose diseases is increasing, it remains a challenge in the case of tick-borne conditions. For example, in 2017, 160 cases of the Brazilian Spotted Fever (BSF, a tick-borne illness) were confirmed, alarming the notifiable diseases information system. Conversely, Brazilian borreliosis and ehrlichiosis do not require notification. Still, an increasing number of cases in humans and dogs have been reported in southeast and northeastern Brazil. Immunological methods applied to human and dog tick-borne diseases (TBD) show low sensitivity and specificity, cross-reactions, and false IgM positivity. Thus, the diagnosis and management of TBD are hampered by the personal tools and indirect markers used. Therefore, specific and rapid methods urgently need to be developed to diagnose the various types of tick-borne bacterial diseases. This review presents a brief historical perspective on the evolution of serological assays and recent advances in diagnostic tests for TBD (ehrlichiosis, BSF, and borreliosis) in humans and dogs, mainly applied in Brazil. Additionally, this review covers the emerging technologies available in diagnosing TBD, including biosensors, and discusses their potential for future use as gold standards in diagnosing these diseases.

Background: Health care-associated infections (HAIs) are a significant public health problem worldwide, favoring multidrug-resistant (MDR) microorganisms. The SARS-CoV-2 infection was negatively associated with the increase in antimicrobial resistance, and the ESKAPE group had the most significant impact on HAIs. The study evaluated the bactericidal effect of a high concentration of O3 gas on some reference and ESKAPE bacteria. Material and Methods: Four standard strains and four clinical or environmental MDR strains were exposed to elevated ozone doses at different concentrations and times. Bacterial inactivation (growth and cultivability) was investigated using colony counts and resazurin as metabolic indicators. Scanning electron microscopy (SEM) was performed. Results: The culture exposure to a high level of O3 inhibited the growth of all bacterial strains tested with a statistically significant reduction in colony count compared to the control group. The cell viability of S. aureus (MRSA) (99.6%) and P. aeruginosa (XDR) (29.2%) was reduced considerably, and SEM showed damage to bacteria after O3 treatment Conclusion: The impact of HAIs can be easily dampened by the widespread use of ozone in ICUs. This product usually degrades into molecular oxygen and has a low toxicity compared to other sanitization products. However, high doses of ozone were able to interfere with the growth of all strains studied, evidencing that ozone-based decontamination approaches may represent the future of hospital cleaning methods.

Background: The coronavirus disease of 2019 (COVID-19) is caused by an infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was recognized in late 2019 and has since spread worldwide, leading to a pandemic with unprecedented health and financial consequences. There remains an enormous demand for new diagnostic methods that can deliver fast, low-cost, and easy-to-use confirmation of a SARS-CoV-2 infection. We have developed an affordable electrochemical biosensor for the rapid detection of serological immunoglobulin G (IgG) antibody in sera against the spike protein. Materials and Methods: A previously identified linear B-cell epitope (EP) specific to the SARS-CoV-2 spike glycoprotein and recognized by IgG in patient sera was selected for the target molecule. After synthesis, the EP was immobilized onto the surface of the working electrode of a commercially available screen-printed electrode (SPE). The capture of SARS-CoV-2-specific IgGs allowed the formation of an immunocomplex that was measured by square-wave voltammetry from its generation of hydroquinone (HQ). Results: An evaluation of the performance of the EP-based biosensor presented a selectivity and specificity for COVID-19 of 93% and 100%, respectively. No cross-reaction was observed to antibodies against other diseases that included Chagas disease, Chikungunya, Leishmaniosis, and Dengue. Differentiation of infected and non-infected individuals was possible even at a high dilution factor that decreased the required sample volumes to a few microliters. Conclusion: The final device proved suitable for diagnosing COVID-19 by assaying actual serum samples, and the results displayed good agreement with the molecular biology diagnoses. The flexibility to conjugate other EPs to SPEs suggests that this technology could be rapidly adapted to diagnose new variants of SARS-CoV-2 or other pathogens.

Intracellular parasites such as Trypanosoma cruzi need to acquire valuable carbon sources from the host cell to replicate. Here, we investigated the energetic metabolism of T. cruzi during metacyclogenesis through the determination of enzymatic activities and quantification by HPLC of glycolytic and Krebs cycle short-chain carboxylic acids. Altered concentrations in pyruvate, acetate, succinate, and glycerate were measured during the growth of epimastigote in the complex medium BHI and their differentiation to trypomastigotes in the chemically defined medium, TAU3AAG. These alterations should represent significant differential metabolic modifications utilized by either form to generate energy. This paper is the first work dealing with the intracellular organic acid concentration measurement in T. cruzi parasites. Although it confirms the previous assumption of the importance of carbohydrate metabolism, it yields an essential improvement in T. cruzi metabolism knowledge.

Background: Angiostrongyliasis, the leading cause universal of eosinophilic meningitis, is an emergent disease due to Angiostrongylus cantonensis (rat lungworm) larvae, transmitted accidentally to humans. The diagnosis of human angiostrongyliasis is based on epidemiologic characteristics, clinical symptoms, medical history, and laboratory findings, particularly hypereosinophilia in blood and cerebrospinal fluid. Thus, the diagnosis is difficult and often confused with those produced by other parasitic diseases. Therefore, the development of a fast and specific diagnostic test for angiostrongyliasis is a challenge mainly due to the lack of specificity of the described tests, and therefore, the characterization of a new target is required. Material and Methods: Using bioinformatics tools, the putative presenilin (PS) protein C7BVX5-1 was characterized structurally and phylogenetically. A peptide microarray approach was employed to identify single and specific epitopes, and tetrameric epitope peptides were synthesized to evaluate their performance in an ELISA-peptide assay. Results: The data showed that the A. cantonensis PS protein presents nine transmembrane domains, the catalytic aspartyl domain [(XD (aa 241) and GLGD (aa 332–335)], between TM6 and TM7 and the absence of the PALP and other characteristics domains of the class A22 and homologous presenilin (PSH). These individualities make it an atypical sub-branch of the PS family, located in a separate subgroup along with the enzyme Haemogonchus contournus and separated from other worm subclasses. Twelve B-linear epitopes were identified by microarray of peptides and validated by ELISA using infected rat sera. In addition, their diagnostic performance was demonstrated by an ELISA-MAP4 peptide. Conclusions: Our data show that the putative AgPS is an atypical multi-pass transmembrane protein and indicate that the protein is an excellent immunological target with two (PsAg3 and PsAg9) A. costarisencis cross-reactive epitopes and eight (PsAg1, PsAg2, PsAg6, PsAg7, PsAg8, PsAg10, PsAg11, PsAg12) apparent unique A. cantonensis epitopes. These epitopes could be used in engineered receptacle proteins to develop a specific immunological diagnostic assay for angiostrongyliasis caused by A. cantonensis.

(1) Background: Disinfection of medical devices designed for clinical use associated or not with the growing area of tissue engineering is an urgent need. However, traditional disinfection methods are not always suitable for some biomaterials, especially those sensitive to chemical, thermal, or radiation. Therefore, the objective of this study was to evaluate the minimal concentration of ozone gas (O3) necessary to control and kill a set of sensitive or multi-resistant Gram-positive and Gram-negative bacteria. The cell viability, membrane permeability, and the levels of reactive intracellular oxygen (ROS) species were also investigated; (2) Material and Methods: Four standard strains and a clinical MDR strain were exposed to low doses of ozone at different concentrations and times. Bacterial inactivation (cultivability, membrane damage) was investigated using colony counts, resazurin as a metabolic indicator, and propidium iodide (PI). A fluorescent probe (H2DCFDA) was used for the ROS analyses; (3) Results: No reduction in the count colony was detected after O3 exposure compared to the control group. However, the cell viability of E. coli (30%), P. aeruginosa (25%), and A. baumannii (15%) was reduced considerably. The bacterial membrane of all strains was not affected by O3 but presented a significant increase of ROS in E. coli (90 ± 14%), P. aeruginosa (62.5 ± 19%), and A. baumanni (52.6 ± 5%); (4) Conclusion: Low doses of ozone were able to interfere in the cell viability of most strains studied, and although it does not cause damage to the bacterial membrane, increased levels of reactive ROS are responsible for causing a detrimental effect in the lipids, proteins, and DNA metabolism.

HSV infections, both type 1 and type 2, are among the most widespread viral diseases affecting people of all ages. Their symptoms could be mild, with cold sores up to 10 days of infection, blindness and encephalitis caused by HSV-1 affecting immunocompetent and immunosuppressed individuals. The severe effects derive from co-evolution with the host, resulting in immune evasion mechanisms, including latency and growing resistance to acyclovir and derivatives. An efficient alternative to controlling the spreading of HSV mutations is the exploitation of new drugs, and the possibility of enhancing their delivery through the encapsulation of drugs into nanoparticles, such as liposomes. In this work, liposomes were loaded with a series of 2-aminomethyl- 3-hydroxy-1,4-naphthoquinones derivatives with n-butyl (compound 1), benzyl (compound 2) and nitrobenzene (compound 3) substituents in the primary amine of naphthoquinone. They were previously identified to have significant inhibitory activity against HSV-1. All of the aminomethylnaphthoquinones derivatives encapsulated in the phosphatidylcholine liposomes were able to control the early and late phases of HSV-1 replication, especially those substituted with the benzyl (compound 2) and nitrobenzene (compound 3), which yields selective index values that are almost nine times more efficient than acyclovir. The growing interest of the industry in topical administration against HSV supports our choice of liposome as a drug carrier of aminomethylnaphthoquinones derivatives for formulations of in vivo pre-clinical assays.

The COVID-19 pandemic has exposed the extent of global connectivity and collective vulnerability to emerging diseases. From its suspected origins in Wuhan, China, it spread to all corners of the world in a matter of months. The absence of high-performance, rapid diagnostic methods that could identify asymptomatic carriers contributed to its worldwide transmission. Serological tests offer numerous benefits compared to other assay platforms to screen large populations. First-generation assays contain targets that represent proteins from SARS-CoV-2. While they could be quickly produced, each actually has a mixture of specific and non-specific epitopes that vary in their reactivity for antibodies. To generate the next generation of the assay, epitopes were identified in three SARS-Cov-2 proteins (S, N, and Orf3a) by SPOT synthesis analysis. After their similarity to other pathogen sequences was analyzed, 11 epitopes outside of the receptor-binding domain (RBD) of the spike protein that showed high reactivity and uniqueness to the virus. These were incorporated into a ß-barrel protein core to create a highly chimeric protein. Another de novo protein was designed that contained only epitopes in the RBD. In-house ELISAs suggest that both multiepitope proteins can serve as targets for high-performance diagnostic tests. Our approach to bioengineer chimeric proteins is highly amenable to other pathogens and immunological uses.

The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggest it contains nine transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT-synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles, and endoplasmic reticulum in parasites by whole-cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a target for the generation of new therapeutics specific to the T. cruzi.

Tropical diseases caused by parasitic infections continue to cause socioeconomic distress worldwide. Among these, Chagas disease has become a great concern because of globalization. Caused by Trypanosoma cruzi, there is an increasing need to discover new, more effective methods to manage infections that minimize disease onset. Antimicrobial peptides represent a possible solution to this challenge. As effector molecules of the innate immune response against pathogens, they are the first line of defense found in all multi-cellular organisms. In amphibians, temporins are a large family of antimicrobial peptides found in skin secretions. Their functional roles and modes of action present unique properties that indicate possible candidates for therapeutic applications. Here, we investigated the trypanocide activity of temporizin and temporizin-1. Temporizin is an artificial, hybrid peptide containing the N-terminal region of temporin A, the pore-forming region of gramicidin and a C-terminus consisting of alternating leucine and lysine. Temporizin-1 is a modification of temporizin with a reduction in the region responsible for insertion into membranes. Their activities were evaluated in a cell permeabilization assay by flow cytometry, an LDH release assay, electron microscopy, an MTT assay and patch clamp experiments. Both temporizin and temporizin-1 demonstrated toxicity against T. cruzi with temporizin displaying slightly more potency. At concentrations up to 100 μg/ ml, both peptides exhibited low toxicity in J774 cells, a macrophage lineage cell line, and no toxicity was observed in mouse primary peritoneal macrophages. In contrast, the peptides showed some toxicity in rat adenoma GH3 cells and Jurkat human lymphoma cells with temporizin-1 displaying lower toxicity. In summary, a shortened form of the hybrid temporizin peptide, temporizin-1, was efficient at killing T. cruzi and it has low toxicity in wild-type mammalian cells. These data suggest that temporizin-1 might be a candidate for Chagas disease therapy.