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A transgenic protein is frequently expressed as different homologous variants in genetically modified crops due to differential processing of targeting peptides or optimization of activity and specificity. The aim of this study was to develop a science-based approach for risk assessment of homologous protein variants using dicamba mono-oxygenase (DMO) as a case study. In this study, DMO expressed in the next-generation dicamba-tolerant maize, sugar beet and soybean crops exhibited up to 27 amino acid sequence differences in the N-terminus. Structure modeling using AlphaFold, ESMFold and OpenFold demonstrates that these small N-terminal extensions lack an ordered secondary structure and do not disrupt the DMO functional structure. Three DMO variants were demonstrated to have equivalent immunoreactivity and functional activity ranging from 214 to 331 nmol/min/mg. Repeated toxicity studies using each DMO variant found no test substance-related adverse effects. These results support that homologous protein variants, which have demonstrated physicochemical and functional equivalence, can leverage existing safety data from one variant without requiring additional de novo safety assessments.

Leishmaniasis is a complex disease caused by infection with different Leishmania parasites. The number of medications used for its treatment is still limited and the discovery of new drugs is a valuable approach. In this context, here we describe the in vitro leishmanicidal activity and the in silico interaction between trypanothione reductase (TryR) and (-)-5-demethoxygrandisin B from the leaves of Virola surinamensis (Rol.) Warb. The compound (-)-5-demethoxygrandisin B was isolated from V. surinamensis leaves, a plant found in the Brazilian Amazon, and it was characterized as (7R,8S,7R,8S)-3,4,5,3,4-pentamethoxy-7,7-epoxylignan. In vitro antileishmanial activity was examined against Leishmania amazonensis, covering both promastigote and intracellular amastigote phases. Cytotoxicity and nitrite production were gauged using BALB/c peritoneal macrophages. Moreover, transmission electron microscopy was applied to probe ultrastructural alterations, and flow cytometry assessed the shifts in the mitochondrial membrane potential. In silico methods such as molecular docking and molecular dynamics assessed the interaction between the most stable configuration of (-)-5-demethoxygrandisin B and TryR from L. infantum (PDB ID 2JK6). As a result, the (-)-5-demethoxygrandisin B was active against promastigote (IC50 7.0 µM) and intracellular amastigote (IC50 26.04 µM) forms of L. amazonensis, with acceptable selectivity indexes. (-)-5-demethoxygrandisin B caused ultrastructural changes in promastigotes, including mitochondrial swelling, altered kDNA patterns, vacuoles, vesicular structures, autophagosomes, and enlarged flagellar pockets. It reduced the mitochondria membrane potential and formed bonds with important residues in the TryR enzyme. The molecular dynamics simulations showed stability and favorable interaction with TryR. The compound targets L. amazonensis mitochondria via TryR enzyme inhibition.

The toxicity of tacrolimus metabolites and their potential pharmacodynamic (PD) interactions with tacrolimus might respectively explain the surprising combination of higher toxicity and lower efficacy of tacrolimus despite normal blood concentrations, described in extensive metabolizers. To evaluate such interactions, we produced tacrolimus metabolites in vitro and characterized them by high resolution mass spectrometry (HRMS, for all) and nuclear magnetic resonance (NMR, for the most abundant, M-I). We quantified tacrolimus metabolites and checked their structure in patient whole blood and peripheral blood mononuclear cells (PBMC). We explored the interactions of M-I with tacrolimus in silico, in vitro and ex vivo. In vitro metabolization produced isoforms of tacrolimus and of its metabolites M-I and M-III, whose HRMS fragmentation suggested an open-ring structure. M-I and M-III open-ring isomers were also observed in patient blood. By contrast, NMR could not detect these open-ring forms. Transplant patients expressing CYP3A5 exhibited higher M-I/TAC ratios in blood and PBMC than non-expressers. Molecular Dynamics simulations showed that: all possible tacrolimus metabolites and isomers bind FKPB12; and the hypothetical open-ring structures induce looser binding between FKBP12 and calcineurins, leading to lower CN inhibition. In vitro, tacrolimus bound FKPB12 with more affinity than purified M-I, and the pool of tacrolimus metabolites and purified M-I had only weak inhibitory activity on IL2 secretion and not at all on NFAT nuclear translocation. M-I showed no competitive effect with tacrolimus on either test. Finally, M-I or the metabolite pool did not significantly interact with tacrolimus MLR suppression, thus eliminating a pharmacodynamic interaction.

ABSTRACT This study aims to verify whether the inequalities by education level found in the population as a whole are also confirmed among those who subscribed to personal insurance and/or open private pension plans in Brazil between 2012 and 2017. This study is a trailblazer in estimating mortality differentials by education level in populations with personal insurance and open private pensions in Brazil. The research contributes to the understanding of socioeconomic factors related to living conditions and, consequently, mortality. The results show that, even in a population that records lower mortality rates and higher life expectancies when compared to the Brazilian population as a whole, education level plays a major role as a protective factor. Population and death data were provided by 23 insurance groups. Through an agreement with the Ministry of Labor and Social Security, the Applied Mathematics Laboratory of the Federal University of Rio de Janeiro (LabMA/UFRJ) provided the list of insured people to this ministry, data was cross-referenced with government information and returned to the LabMA/UFRJ with identification of education level, when available, as well as indication of deceased, completing company records. Mortality tables were then drawn up for the insured population by education level. To obtain smoothed tables, Heligman and Pollard’s law was applied using a Bayesian approach, via Markov Chain Monte Carlo (MCMC) simulation. In the period from 2012 to 2017, among Brazilians who took advantage of personal insurance and open supplementary pensions, men with a high education level had, at the age of 60, a life expectancy of 5.6 and 6.6 years greater than those with a lower education level, in mortality and survival coverage, respectively. In the case of women, the comparison between the most educated and the least educated shows a difference of 2.7 years in mortality coverage and 5.4 years in survival coverage.

Erythropoietic and hemoglobin DNA transcriptional activities were analyzed in the diploid and the tetraploid Odontophrynus americanus. Flow cytometric analyses of DNA, RNA and mitochondrial contents showed increased genic activity in both diploid and tetraploid animals during erythropoiesis in vivo elicited by pretreatment phenylhydrazine. Generally, higher values were seen in immature tetraploid erythroid cells. On the 10th day of recovery from anemia, large amounts of messenger RNA were found in both specimens. Based on the mitochondrial content, the tetraploid cells had more intense energy metabolism than the diploid cells. Diploid O. americanus had about three times more erythroid cells than tetraploid specimens, indicating that there were differences in the regulatory mechanisms of erythroid cells. Hematological parameters showed that tetraploid cells had 30% more hemoglobin than the diploid, suggesting a regulatory mechanism of hemoglobin synthesis at the transcriptional level. Cytoplasmic inclusions resembling Heinz bodies were found in both types of cells. In the tetraploid cells they were previously found associated with RNA or RNP, suggesting that other regulatory system which controls the accumulation of nontranslated RNA transcribed in excess must be present. These differences at the physiological and molecular levels during erythropoiesis reinforce the hypothesis that speciation is occurring between diploid and tetraploid O. americanus.

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-g) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-g and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-g and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response

Abstract The biology underlying proton minibeam radiation therapy (pMBRT) is not fully understood. Here we aim to elucidate the biological effects of pMBRT using Fourier Transform Infrared Microspectroscopy (FTIRM). In vitro (CTX-TNA2 astrocytes and F98 glioma rat cell lines) and in vivo (healthy and F98-bearing Fischer rats) irradiations were conducted, with conventional proton radiotherapy and pMBRT. FTIRM measurements were performed at ALBA Synchrotron, and multivariate data analysis methods were employed to assess spectral differences between irradiation configurations and doses. For astrocytes, the spectral regions related to proteins and nucleic acids were highly affected by conventional irradiations and the high-dose regions of pMBRT, suggesting important modifications on these biomolecules. For glioma, pMBRT had a great effect on the nucleic acids and carbohydrates. In animals, conventional radiotherapy had a remarkable impact on the proteins and nucleic acids of healthy rats; analysis of tumour regions in glioma-bearing rats suggested major nucleic acid modifications due to pMBRT.

Porcine respiratory disease complex represents a major challenge for the swine industry, with swine influenza A virus (swIAV) and porcine reproductive and respiratory syndrome virus (PRRSV) being major contributors. Epidemiological studies have confirmed the co-circulation of these viruses in pig herds, making swIAV-PRRSV co-infections expected. A couple of in vivo co-infection studies have reported replication interferences between these two viruses. Herein, using a reductionist in vitro model, we investigated the potential mechanisms of these in vivo interferences. We first examined the impact of swIAV on porcine alveolar macrophages (AMs) and its effects on AMs co-infection by PRRSV. This was done either in monoculture or in co-culture with respiratory tracheal epithelial cells to represent the complexity of the interactions between the viruses and their respective target cells (epithelial cells for swIAV and AMs for PRRSV). AMs were obtained either from conventional or specific pathogen-free (SPF) pigs. SwIAV replication was abortive in AMs, inducing cell death at high multiplicity of infections. In AMs from three out of four conventional animals, swIAV showed no impact on PRRSV replication. However, inhibition of PRRSV multiplication was observed in AMs from one animal, accompanied by an early increase in the expression of interferon (IFN)-I and IFN-stimulated genes. In AMs from six SPF pigs, swIAV inhibited PRRSV replication in all animals, with an early induction of antiviral genes. Co-culture experiments involving tracheal epithelial cells and AMs from either SPF or conventional pigs all showed swIAV-induced inhibition of PRRSV replication, together with early induction of antiviral genes. These findings highlight the complex interactions between swIAV and PRRSV in porcine AMs, and would suggest a role of host factors, such as sanitary status, in modulating viral propagation. Our co-culture experiments demonstrated that swIAV inhibits PRRSV replication more effectively in the presence of respiratory tracheal epithelial cells, suggesting a synergistic antiviral response between AMs and epithelial cells, consistent with in vivo experiments.

Abstract Background Radiation-induced neurocognitive dysfunction is a major adverse effect of brain radiation therapy and has specific relevance in pediatric oncology, where serious cognitive deficits have been reported in survivors of pediatric brain tumors. Moreover, many pediatric patients receive proton therapy under general anesthesia or sedation to guarantee precise ballistics with a high oxygen content for safety. The present study addresses the relevant question of the potential effect of supplemental oxygen administered during anesthesia on normal tissue toxicity and investigates the anti-tumor immune response generated following conventional and FLASH proton therapy. Methods Rats (Fischer 344) were cranially irradiated with a single high dose of proton therapy (15 Gy or 25 Gy) using FLASH dose rate proton irradiation (257 ± 2 Gy/s) or conventional dose rate proton irradiation (4 ± 0.02 Gy/s), and the toxicities in the normal tissue were examined by histological, cytometric and behavioral analysis. Glioblastoma-bearing rats were irradiated in the same manner and tumor-infiltrating leukocytes were quantified by flow cytometry. Results Our findings indicate that supplemental oxygen has an adverse impact on both functional and anatomical evaluations of normal brain following conventional and FLASH proton therapy. In addition, oxygen supplementation in anesthesia is particularly detrimental for anti-tumor immune response by preventing a strong immune cell infiltration into tumoral tissues following conventional proton therapy. Conclusions These results demonstrate the need to further optimize anesthesia protocols used in radiotherapy with the goal of preserving normal tissues and achieving tumor control, specifically in combination with immunotherapy agents.

Abstract Background Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant. Results Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities. Conclusions Based on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.

Resumo Objetivo Analisar o conceito de “autogestão ineficaz do linfedema” em mulheres mastectomizadas. Métodos Trata-se de uma análise do conceito proposto por Walker e Avant operacionalizada por meio de uma revisão integrativa da literatura, organizada em oito etapas e baseada nas seguintes questões norteadoras: Qual é a definição de autogestão ineficaz em mulheres mastectomizadas? Quais os antecedentes, atributos e consequências da autogestão ineficaz em mulheres mastectomizadas? Como a autogestão ineficaz de linfedema tem sido definida no contexto de mulheres que passaram por mastectomia? A busca de artigos foi realizada em cinco bases de dados eletrônicas, sem limite de data, no período de julho de 2022 a fevereiro de 2023. Resultados Foram identificados 30 antecedentes e 19 consequentes; os mais frequentes entre eles foram respectivamente os seguintes: falta de apoio familiar/social e diminuição da qualidade de vida. Os atributos definidores mais frequentemente identificados foram os seguintes: edema, inchaço no braço, sensação de peso no membro, dor, dormência e diminuição da função do membro. Foram ainda elaborados os casos modelo e contrário para auxiliar na decisão sobre o uso do conceito. Conclusão Os conceitos resultantes da análise contribuem para clarificar os termos e o desenvolvimento da linguagem na enfermagem, devendo ser validados por juízes e prática clínica para melhor aplicação na oncologia clínica.

Abstract:

Modeling of tsunami waves generated by subaerial landslides is important to provide accurate hazard and risk assessments in coastal areas. We perform small-scale laboratory experiments where a tsunami-like wave is generated by the gravity-driven collapse of a subaerial granular column into water. We show that the maximal amplitude reached near-shore by the generated wave in our experiments is linked to the instantaneous immersed volume of grains and to the ultimate immersed deposit. Despite the differences in scale and geometry between our small-scale experiments and the larger-scale geophysical events, a rather good agreement is found between the experimental law and the field data. This approach offers an easy way to estimate the amplitude of paleo-tsunamis.

Abstract This article presents the Brazilian private insurance market’s actuarial life tables, BR- EMS 2021. Using Bayesian inference on the parameters of the Heligman- Pollard law of mortality and data from 23 insurance groups over 15 years, totaling 3.5 billion registers, the data were corrected through a two hidden-layer neural network. The resulting tables show that the insured population exhibits lower mortality rates than the general Brazilian population, even lower than the national populations of well-developed countries such as the USA. Moreover, besides the expected gender gap in mortality rates, there is a clear distance between the death and survivorship insurance coverage groups. Likewise, the insured population characteristics mitigate well-known regional structural discrepancies in the Brazilian population, indicating that being part of the selected population of insured individuals is thus associated with a more effective protection against death than other outstanding factors such as geographic region of residence.

Gastrointestinal parasitism is a major health and welfare problem in ruminants. Synthetic chemical anthelmintic drugs have led to the emergence of resistance in gastrointestinal strongyles, inducing the search for alternatives to control the infections that affect ruminants. The objective of this work was to evaluate the anthelmintic potential of plant extracts against Haemonchus contortus Rudolphi. Three plants of the Guadeloupean biodiversity, Momordica charantia L., Carica papaya L. and Sargassum spp., were selected based on their high polyphenolic content and natural abundance. The phytochemistry of plants was explored, a biological assay against the parasite H. contortus was carried out, and several hypotheses about the way of action were proposed by an innovative electrochemical screening method.

Simple Summary: Diffuse large B-cell lymphoma (DLBCL) is a very common and aggressive form of blood cancer. Despite therapeutic advances, ~40% of DLBCL patients will die. Metabolic reprogramming is a characteristic of cancer cells and a new potential therapeutic target. Our research focuses on developing strategies by targeting metabolic vulnerabilities to improve clinical outcomes for DLBCL patients with therapeutic impasses. Here, we report that combining two antimetabolic drugs, namely metformin and L-asparaginase, induced massive DLBCL cell killing compared to each drug alone. Further, we deciphered how this novel drug combination impacts critical metabolic and oncogenic pathways. Finally, we demonstrated the clinical benefit of this optimized antimetabolic drug treatment for DLBCL patients. Altogether, our data provide a framework for the use of an optimized combination of antimetabolic drugs to overcome resistance in DLBCL patients. Background/Objectives: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, characterized by aggressive and heterogeneous tumors originating from B-cells. Especially in patients with relapsed or refractory (R/R) disease, DLBCL remains a challenging cancer to treat. Metabolic reprogramming is a hallmark of malignant cells. Our research focuses on developing strategies to enhance clinical outcomes for R/R DLBCL patients by targeting metabolic vulnerabilities. Methods: We investigated the effects of combining metformin and L-asparaginase, two FDA-approved antimetabolic drugs, on DLBCL cell metabolism and survival. Nuclear magnetic resonance (NMR) spectroscopy was employed to assess metabolic disturbances induced by the drug combination. The impact on lipid metabolism, glycolysis, glutaminolysis, the tricarboxylic acid (TCA) cycle, and antioxidant responses was examined. Induction of apoptosis was evaluated by FACS analysis. Results: The combination of metformin and L-asparaginase strongly sensitized DLBCL cells to apoptosis, independently of their oxidative phosphorylation (OxPhos) or BCR/glycolytic status. NMR spectroscopy revealed that this combination induces broader metabolic disturbances than either drug alone. It disrupts lipid metabolism by altering levels of phospholipids, cholesterol, and fatty acids. Additionally, it counteracts the pro-glycolytic effect of metformin, decreases glycolysis, and reduces glutaminolysis. It also affects the TCA cycle and antioxidant responses, critical for cellular energy production and redox balance. Furthermore, this combination interferes with two key cancer survival pathways, mTORC1 and MAPK signaling. Importantly, proof of principle for its beneficial effect was demonstrated in DLBCL patients. Conclusions: Combining metformin and L-asparaginase affects DLBCL cell survival by targeting multiple metabolic pathways and may represent a novel therapeutic approach for R/R DLBCL patients. [ABSTRACT FROM AUTHOR]

Radioresistant tumours remain complex to manage with current radiotherapy (RT) techniques. Heavy ion beams were proposed for their treatment given their advantageous radiobiological properties. However, previous studies with patients resulted in serious adverse effects in the surrounding healthy tissues. Heavy ion RT could therefore benefit from the tissue-sparing effects of minibeam radiation therapy (MBRT). To investigate the potential of this combination, here we assessed the biochemical response to neon MBRT (NeMBRT) through synchrotron-based Fourier transform infrared microspectroscopy (SR-FTIRM). Healthy (BJ) and tumour (B16-F10) cell lines were subjected to seamless (broad beam) neon RT (NeBB) and NeMBRT at HIMAC. SR-FTIRM measurements were conducted at the MIRAS beamline of ALBA Synchrotron. Principal component analysis (PCA) permitted to assess the biochemical effects after the irradiations and 24 hours post-irradiation for the different RT modalities and doses. For the healthy cells, NeMBRT resulted in the most dissimilar spectral signatures from non-irradiated cells early after irradiations, mainly due to protein conformational modifications. Nevertheless, most of the damage appeared to recover one day post-RT; conversely, protein- and nucleic acid-related IR bands were strongly affected by NeBB 24 hours after treatment, suggesting superior oxidative damage and nucleic acid degradation. Tumour cells appeared to be less sensitive to NeBB than to NeMBRT shortly after RT. Still, after one day, both NeBB and the high-dose NeMBRT regions yielded important spectral modifications, suggestive of cell death processes, protein oxidation or oxidative stress. Lipid-associated spectral changes, especially due to the NeBB and NeMBRT peak groups for the tumour cell line, were consistent with reactive oxygen species attacks. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis is a neglected tropical disease caused, in Brazil, mainly by Leishmania braziliensis, which is a protozoan transmitted during the blood feeding of infected female sandflies. To control leishmaniasis, the participation of CD4+ Th1 cells together with macrophages, neutrophils, and other peripheral blood cells, including platelets, is necessary. These anuclear fragments, when activated, produce microvesicles (MVs) that can reach locations outside the blood, carrying molecules responsible for activating pro-inflammatory responses and antigen presentation. Using flow cytometry, this current study evaluated the frequency and concentration of platelet-derived MVs (pMVs) in plasma samples obtained from patients in the acute phase and undergoing treatment, as well as from healthy volunteers. Our results revealed a higher frequency and concentration of pMVs in the plasma of patients with acute CL when compared to all other groups studied. These results highlight the impact of pMVs in modulating the immune response of CL patients, correlating their higher concentrations and frequencies in CL-patient plasmas, with the acute inflammatory status of the disease and their reduction with beneficial results of systemic treatment with antimony. This knowledge is essential to define potential treatment protocols, as well as highlight pMVs as biomarkers for the different clinical stages of CL.