Exploring "dark-matter" protein folds using deep learning.

De novo protein design explores uncharted sequence and structure space to generate novel proteins not sampled by evolution. A main challenge in de novo design involves crafting "designable" structural templates to guide the sequence searches toward adopting target structures. We present a convolutional variational autoencoder that learns patterns of protein structure, dubbed Genesis. We coupled Genesis with trRosetta to design sequences for a set of protein folds and found that Genesis is capable of reconstructing native-like distance and angle distributions for five native folds and three novel, the so-called "dark-matter" folds as a demonstration of generalizability. We used a high-throughput assay to characterize the stability of the designs through protease resistance, obtaining encouraging success rates for folded proteins. Genesis enables exploration of the protein fold space within minutes, unrestricted by protein topologies. Our approach addresses the backbone designability problem, showing that small neural networks can efficiently learn structural patterns in proteins. A record of this paper's transparent peer review process is included in the supplemental information.

Structures of the Foamy virus fusion protein reveal an unexpected link with the F protein of paramyxo- and pneumoviruses.

Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope (Env) glycoprotein drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report the cryo-electron microscopy structures of the FV Env ectodomain in the pre- and post-fusion states, which unexpectedly demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the viral fusogens. We describe the structural features that are unique to the FV Env and propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive membrane fusion and viral entry. The structural knowledge on the FV Env now provides a framework for functional investigations, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors.

Dual ON/OFF-switch chimeric antigen receptor controlled by two clinically approved drugs.

The ability to remotely control the activity of chimeric antigen receptors (CARs) with small molecules can improve the safety and efficacy of gene-modified T cells. Split ON- or OFF-switch CARs involve the dissociation of tumor-antigen binding from T cell activation (i.e., CD3ζ) on the receptor (R-) and signaling (S-) chains, respectively, that either associate or are disrupted in the presence of a small molecule. Here, we have developed an inducible (i)ON-CAR comprising the anti-apoptotic B cell lymphoma protein 2 protein in the ectodomain of both chains which associate in the presence of venetoclax. We showed that inducible ON (iON)-CAR T cells respond to target tumors cells in the presence of venetoclax or the BH3 mimetic navitoclax in a dose-dependent manner, while there is no impact of the drugs on equivalent second generation-CAR T cells. Within 48 h of venetoclax withdrawal, iON-CAR T cells lose the ability to respond to target tumor cells in vitro as evaluated by Interferon-gamma (IFNγ) production, and they are reliant upon the presence of venetoclax for in vivo activity. Finally, by fusing a degron sequence to the endodomain of the iON-CAR S-chain we generated an all-in-one ON/OFF-switch CAR, the iONØ-CAR, down-regulated by lenalidomide within 4 to 6 for functionally inactive T cells (no IFNγ production) within 24 h. We propose that our remote-control CAR designs can reduce toxicity in the clinic. Moreover, the periodic rest of iON and iONØ-CAR T cells may alleviate exhaustion and hence augment persistence and long-term tumor control in patients.

Comparative life-history strategies in three Lebiasinidae (Characiformes) in a Rio Negro tributary, Brazilian Amazon.

Reproductive traits co-evolve and form successful life-history strategies adapted to the biology and environment of a particular taxon, maximizing offspring and species survival chances, therefore studies investigating differences in adaptive traits across different environments can enhance our understanding of the natural selection process and evolution. Herein, we address whether the reproductive strategies of phylogenetically closely related fishes are influenced by habitat predictability, using three species of the Lebiasinidae family as models. The predominance of larger and mature individuals during the flood season, with high waters characterized by smaller immature individuals, suggests a seasonal reproductive strategy for Nannostomus trifasciatus. Copella callolepis, which inhabits both habitats, also showed a single reproductive peak. However, compared to N. trifasciatus, this species displayed late spawning, restricted to the flood season, as indicated by the higher abundance of larger and mature individuals during this period and the presence of smaller (juveniles) and spawned individuals in the following season. The reproductive tactics observed in N. marginatus differed significantly from the single reproductive peak of the other species, as two reproductive peaks were observed: one during the flood season and another during the low water season. In conclusion, our study demonstrates that the environment strongly influences reproductive strategies for lebiasinids. N. marginatus, restricted to small water bodies, exhibited an opportunistic reproductive strategy, whereas the species inhabiting main rivers, N. trifasciatus and C. callolepis, exhibited a more seasonal strategy.

An evolved artificial radical cyclase enables the construction of bicyclic terpenoid scaffolds via an H-atom transfer pathway.

While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Here we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyses a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co cofactor is confirmed by crystallography. A time course experiment reveals a cascade reaction catalysed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibits tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.

Trend in mortality from mental and behavioral disorders due to alcohol use in Brazil, 2010-2021.

OBJECTIVE: To analyze the trend in mortality from mental and behavioral disorders due to alcohol use in Brazil, 2010-2021. METHODS: This was an time series study using Mortality Information System data. Annual percentage change (APC) and 95% confidence intervals (95% CI) were calculated using Prais-Winsten linear regression. RESULTS: Mortality showed a stationary trend for Brazil as a whole (APC = 0.6; 95%CI -4.2;3.0), a falling trend in individuals aged 20-29 years in the South (APC = -7.4; 95%CI -10.0;-4.3) and Northeast (APC = -3.4; 95%CI -6.4;-0.4) regions, in people aged 30-39 in the Midwest region (APC = -3,8; 95%CI -7.4;-0.1) and 40-49 in the South (APC = -2.1; 95%CI -3.8;-0.4), North (APC = -3.1; 95%CI -5.7;-0.5) and Midwest (APC = -2.9; 95%CI -5.5;-0.3) regions. CONCLUSION: Mortality from mental and behavioral disorders due to alcohol use showed a stationary trend nationally and a falling trend in some age groups regionally.

Isolation and In Vitro Biological Evaluation of Triterpenes from Salacia grandifolia Leaves.

Salacia grandifolia is naturally found in the Atlantic Forest regions of Brazil. Despite the pharmacological potential of plants from the Salacia genus, phytochemical studies on this species have not been reported in literature. A new triterpene, 28-hydroxyfriedelane-3,15-dione (1), and seven known compounds (friedelan-3-one (2), friedelan-3ß-ol (3), friedelane-3,15-dione (4), 15α-hydroxyfriedelan-3-one (5), 28-hydroxyfriedelan-3-one (6), 30-hydroxyfriedelan-3-one (7), and 29-hydroxyfriedelan-3-one (8)) were obtained from the hexane extract of Salacia grandifolia leaves. These isolated compounds and three extracts, hexane (EH), chloroform (EC), and ethyl acetate (EAE), were assessed for their potential biological activities, which consisted in the evaluation of antiviral activity against a murine coronavirus, mouse hepatitis virus 3 (MHV-3), antibacterial activity against the susceptible and methicillin-resistant Staphylococcus aureus (MRSA), and antileukemia activity against the THP-1 and K-562 cell lines. The extracts EH and EAE along with the triterpenes 1 and 6 exhibited moderate to high antiviral activity, with emphasis on 6, which presented an EC50 value of 2.9 ± 0.3 µM. None of the compounds presented antibacterial activity against the tested strains. The evaluated compounds 1, 4, 6 and 7 exhibited low cytotoxic activity against the tested leukemia cell lines. Taken together, this study comprises an overview for the potential of the Salacia grandifolia biological activities, including a new isolated triterpene.

Pharmacotherapeutic Considerations on Telomere Biology: The Positive Effect of Pharmacologically Active Substances on Telomere Length.

Telomeres are part of chromatin structures containing repeated DNA sequences, which function as protective caps at the ends of chromosomes and prevent DNA degradation and recombination, thus ensuring the integrity of the genome. While telomere length (TL) can be genetically inherited, TL shortening has been associated with ageing and multiple xenobiotics and bioactive substances. TL has been characterised as a reliable biomarker for the predisposition to developing chronic pathologies and their progression. This narrative review aims to provide arguments in favour of including TL measurements in a complex prognostic and diagnostic panel of chronic pathologies and the importance of assessing the effect of different pharmacologically active molecules on the biology of telomeres. Medicines used in the management of cardiovascular diseases, diabetes, schizophrenia, hormone replacement therapy at menopause, danazol, melatonin, and probiotics have been studied for their positive protective effects against TL shortening. All these classes of drugs are analysed in the present review, with a particular focus on the molecular mechanisms involved.

Computational design of soluble and functional membrane protein analogues.

De novo design of complex protein folds using solely computational means remains a substantial challenge1. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors2, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.

Leptospirosis as a cause of infertility in Uruguayan beef cattle.

Leptospirosis, caused by pathogenic spirochetes of the genus Leptospira spp., is a globally significant zoonotic disease that affects humans and animals. In cattle, leptospirosis is associated not only with overt clinical manifestations but also with reproductive diseases, including infertility. This study assesses the potential correlation between leptospirosis and infertility in Uruguayan beef cattle. A case-control study involved 31 beef herds with no prior history of Leptospira vaccination. In each herd, veterinarians identified 10 non-pregnant (cases) and 25 pregnant cows (controls) using ultrasound, and blood and urine samples were collected from each cow. Serological diagnosis was performed using the Microscopic Agglutination Test (MAT), and quantitative PCR (qPCR) was used to assess Leptospira excretion. Additionally, antibodies against bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis (IBR) were tested. The results demonstrated an association between seropositivity to the Sejroe serogroup (cut-off 1:200) and infertility in cattle (OR=1.31; p-value=0.06). Furthermore, the level of Leptospira excretion (qPCR) in urine was associated with increased infertility risk, with cows excreting over 100 copies per mL of urine having the highest odds of infertility (OR=2.34; p-value<0.01). This study suggests a potential association between leptospirosis and infertility in Uruguayan beef cattle, emphasizing the importance of both serological and molecular diagnostics for assessing reproductive health in cattle herds. Future research should explore the impact of Leptospira serogroups on other reproductive disorders in cattle.

Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.

Stability testing of iomeprol and iopamidol formulations subjected to X-ray radiation.

INTRODUCTION: Exposure of iodinated contrast media (ICM) to X-rays is not uncommon, as contrast media are often stored in close proximity to radiological equipment. However, the interaction between X-rays and ICM is not widely investigated in literature. The present study aims to investigate the chemical stability of iomeprol and iopamidol, two commercial iodinated ICM commonly used in diagnostic imaging, under X-rays exposure. METHODS: Different formulations of iopamidol and iomeprol (iodine concentration 9 to 400 mgI/mL, volume 50-500 mL) were exposed to three different conditions of X-ray irradiation: i) 1 month storage in CT room (≈5-15 mGy); (ii) low-dose protocol (≈10 mGy); (ii) stressed protocol (≈100 mGy). Unexposed and exposed solutions were characterized by high-performance liquid chromatography in terms of concentration of active pharmaceutical ingredient (API), iodine species and by products. In addition, appearance and colour of the solutions were inspected and pH measured. RESULTS: API concentrations, appearance, colour and pH of the exposed formulations remained unaffected by X-rays. Measured concentrations of iodine species and by products were observed well within the acceptability criteria, i.e. values turned out to be lower than specifications limits established by the manufacturer, considering both release and shelf-life values. CONCLUSIONS: Up to 100 mGy X-ray exposure did not induce any alteration of iomeprol and iopamidol formulation, nor a detectable increase in the concentration of iodine species or by-products. IMPLICATIONS FOR PRACTICE: Our study strengthens the hypothesis that ICM are stable under X-rays exposure up to 100 mGy.

The Phenomenon of Antiretroviral Drug Resistance in the Context of Human Immunodeficiency Virus Treatment: Dynamic and Ever Evolving Subject Matter.

Human immunodeficiency virus (HIV) is a significant global health issue that affects a substantial number of individuals across the globe, with a total of 39 million individuals living with HIV/AIDS. ART has resulted in a reduction in HIV-related mortality. Nevertheless, the issue of medication resistance is a significant obstacle in the management of HIV/AIDS. The unique genetic composition of HIV enables it to undergo rapid mutations and adapt, leading to the emergence of drug-resistant forms. The development of drug resistance can be attributed to various circumstances, including noncompliance with treatment regimens, insufficient dosage, interactions between drugs, viral mutations, preexposure prophylactics, and transmission from mother to child. It is therefore essential to comprehend the molecular components of HIV and the mechanisms of antiretroviral medications to devise efficacious treatment options for HIV/AIDS.

Dietary Influence on Drug Efficacy: A Comprehensive Review of Ketogenic Diet-Pharmacotherapy Interactions.

It is widely acknowledged that the ketogenic diet (KD) has positive physiological effects as well as therapeutic benefits, particularly in the treatment of chronic diseases. Maintaining nutritional ketosis is of utmost importance in the KD, as it provides numerous health advantages such as an enhanced lipid profile, heightened insulin sensitivity, decreased blood glucose levels, and the modulation of diverse neurotransmitters. Nevertheless, the integration of the KD with pharmacotherapeutic regimens necessitates careful consideration. Due to changes in their absorption, distribution, metabolism, or elimination, the KD can impact the pharmacokinetics of various medications, including anti-diabetic, anti-epileptic, and cardiovascular drugs. Furthermore, the KD, which is characterised by the intake of meals rich in fats, has the potential to impact the pharmacokinetics of specific medications with high lipophilicity, hence enhancing their absorption and bioavailability. However, the pharmacodynamic aspects of the KD, in conjunction with various pharmaceutical interventions, can provide either advantageous or detrimental synergistic outcomes. Therefore, it is important to consider the pharmacokinetic and pharmacodynamic interactions that may arise between the KD and various drugs. This assessment is essential not only for ensuring patients' compliance with treatment but also for optimising the overall therapeutic outcome, particularly by mitigating adverse reactions. This highlights the significance and necessity of tailoring pharmacological and dietetic therapies in order to enhance the effectiveness and safety of this comprehensive approach to managing chronic diseases.

Evaluation of Neuro-Hormonal Dynamics after the Administration of Probiotic Microbial Strains in a Murine Model of Hyperthyroidism.

The microbiota-gut-brain axis has received increasing attention in recent years through its bidirectional communication system, governed by the ability of gut microorganisms to generate and regulate a wide range of neurotransmitters in the host body. In this research, we delve into the intricate area of microbial endocrinology by exploring the dynamic oscillations in neurotransmitter levels within plasma and brain samples. Our experimental model involved inducing hyperthyroidism in mice after a "probiotic load" timeframe using two strains of probiotics (Lactobacillus acidophilus, Saccharomyces boulardii, and their combination). These probiotic interventions continued throughout the experiment and were intended to uncover potential modulatory effects on neurotransmitter levels and discern if certain probiotic strains exhibit any protection from hyperthyroidism. Moreover, we aimed to outline the eventual connections between the gut microbiota and the hypothalamus-pituitary-thyroid axis. As our study reveals, there are significant fluctuations in crucial neurotransmitters within the hyperthyroidism model, related to the specific probiotic strain or combination. These findings could support future therapeutic approaches, help healthcare professionals choose between different probiotic therapies, and also allow us proceed with caution when administering such treatments, depending on the health status of hyperthyroid patients.

Opportunities and challenges in design and optimization of protein function.

The field of protein design has made remarkable progress over the past decade. Historically, the low reliability of purely structure-based design methods limited their application, but recent strategies that combine structure-based and sequence-based calculations, as well as machine learning tools, have dramatically improved protein engineering and design. In this Review, we discuss how these methods have enabled the design of increasingly complex structures and therapeutically relevant activities. Additionally, protein optimization methods have improved the stability and activity of complex eukaryotic proteins. Thanks to their increased reliability, computational design methods have been applied to improve therapeutics and enzymes for green chemistry and have generated vaccine antigens, antivirals and drug-delivery nano-vehicles. Moreover, the high success of design methods reflects an increased understanding of basic rules that govern the relationships among protein sequence, structure and function. However, de novo design is still limited mostly to α-helix bundles, restricting its potential to generate sophisticated enzymes and diverse protein and small-molecule binders. Designing complex protein structures is a challenging but necessary next step if we are to realize our objective of generating new-to-nature activities.

Computational design of soluble functional analogues of integral membrane proteins.

De novo design of complex protein folds using solely computational means remains a significant challenge. Here, we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from GPCRs, are not found in the soluble proteome and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses reveal high thermal stability of the designs and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, standing as a proof-of-concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.

An atlas of protein homo-oligomerization across domains of life.

Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes.

Trend in mortality from mental and behavioral disorders due to alcohol use in Brazil, 2010-2021 / Tendencias de mortalidad por trastornos mentales y comportamentales relacionados con el consumo de alcohol en Brasil: 2010-2021 / Tendência da mortalidade por transtornos mentais e comportamentais devidos ao uso de álcool no Brasil, 2010-2021

Abstract Objective: To analyze the trend in mortality from mental and behavioral disorders due to alcohol use in Brazil, 2010-2021. Methods: This was an time series study using Mortality Information System data. Annual percentage change (APC) and 95% confidence intervals (95% CI) were calculated using Prais-Winsten linear regression. Results: Mortality showed a stationary trend for Brazil as a whole (APC = 0.6; 95%CI -4.2;3.0), a falling trend in individuals aged 20-29 years in the South (APC = -7.4; 95%CI -10.0;-4.3) and Northeast (APC = -3.4; 95%CI -6.4;-0.4) regions, in people aged 30-39 in the Midwest region (APC = -3,8; 95%CI -7.4;-0.1) and 40-49 in the South (APC = -2.1; 95%CI -3.8;-0.4), North (APC = -3.1; 95%CI -5.7;-0.5) and Midwest (APC = -2.9; 95%CI -5.5;-0.3) regions. Conclusion: Mortality from mental and behavioral disorders due to alcohol use showed a stationary trend nationally and a falling trend in some age groups regionally.

Resumen Objetivo: Analizar la tendencia de mortalidad por trastornos mentales y del comportamiento en decorrencia del consumo de alcohol en Brasil en el período 2010-2021. Métodos: Estudio de series de tiempo ecológicas, con datos del Sistema de Información de Mortalidad; el cambio porcentual anual (APV) y los respectivos intervalos de confianza del 95% (IC95%) se calcularon mediante regresión lineal de Prais-Winsten. Resultados: Hubo una tendencia estacionaria en la mortalidad en Brasil en su conjunto (VPA = 0,6; IC95% -4,2;3,0), una tendencia decreciente en individuos de 20 a 29 años en las regiones del Sur (VPA = -7,4; IC95% -10,0;-4,3) y Noreste (VPA = -3,4; IC95% -6,4;-0,4), en personas de 30 a 39 años en el Centro-Oeste (VPA = -3,8; IC95% -7,4;-0,1) y en adultos de 40 a 49 años en el Sur (VPA = -2,1; IC95% -3,8;-0,4), Norte (VPA = -3,1; IC95% -5,7;-0,5) y Centro-Oeste (VPA = - 2,9; IC95% -5,5;-0,3). Conclusión: La mortalidad por trastornos mentales y del comportamiento a causa del consumo de alcohol presentó una tendencia estacionaria en Brasil y una tendencia decreciente en algunos grupos etarios.

Resumo Objetivo: Analisar a tendência da mortalidade por transtornos mentais e comportamentais devidos ao uso de álcool no Brasil em 2010-2021. Métodos: Estudo de séries temporais, com dados do Sistema de Informação sobre Mortalidade. A variação percentual anual (VPA) e respectivos intervalos de confiança de 95% (IC95%) foram calculados por regressão linear de Prais-Winsten. Resultados: Houve tendência estacionária na mortalidade no Brasil como um todo (VPA = 0,6; IC95% -4,2;3,0), tendência decrescente em indivíduos de 20-29 anos nas regiões Sul (VPA = -7,4; IC95% -10,0;-4,3) e Nordeste (VPA = -3,4; IC95% -6,4;-0,4), em pessoas de 30-39 anos no Centro-Oeste (VPA = -3,8; IC95% -7,4;-0,1) e naqueles com 40-49 anos nas regiões Sul (VPA = -2,1; IC95% -3,8;-0,4), Norte (VPA = -3,1; IC95% -5,7;-0,5) e Centro-Oeste (VPA = -2,9; IC95% -5,5;-0,3). Conclusão: A mortalidade por transtornos mentais e comportamentais devidos ao uso de álcool apresentou tendência estacionária no Brasil e decrescente em algumas faixas etárias.