De novo design of high-affinity binders of bioactive helical peptides.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

De novo design of protein structure and function with RFdiffusion.

There has been considerable recent progress in designing new proteins using deep-learning methods1-9. Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.

Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains.

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.

Cardiac rehabilitation in elderly myocardial infarction survivors: focus on circulatory power.

Exercise-based cardiac rehabilitation (EBCR) is paramount after an acute myocardial infarction (AMI). Older individuals have been reported as having a worse prognosis after an AMI, and some series have reported differences in the functional response to EBCR. The peak circulatory power (CP), a non-invasive parameter, has been described as a surrogate for the cardiac power, showing promising results as a comprehensive measure of the cardiovascular response. Whilst this, data concerning the impact of EBCR on CP, particularly among elderly individuals, remains elusive. To address this issue, an observational, retrospective study including all patients admitted due to an AMI who completed a phase II EBCR programme between 11/2012 and 4/2017, was conducted, with CP being analysed by a symptom-limited cardiopulmonary exercise test. A total of 379 patients, 30% aged ≥65 years-old, were included. CP significantly improved after the EBCR programme (in all patients, as well as in both subgroups). Older patients presented lower CP than their younger counterparts at the beginning and the end of the programme, while presenting smaller improvements (122 ± 540 vs 293 ± 638 mmHg mL/kg/min, p = 0.013). This was maintained after adjusting for several potential confounding factors. A contemporary ECBR programme was associated with significant improvements in CP among AMI patients. Though those aged ≥65 years-old presented smaller improvements in CP than younger individuals, these still presented significant increases in this parameter. These results highlight the importance of EBCR in this challenging higher risk group of patients.

Caspase activity in post mortem muscle and its relation to cattle handling practices.

BACKGROUND: Animal handling practices are one of the factors majorly affecting animal metabolism prior to slaughter. This phenomenon increases the occurrence of meat quality defects such as dark cutting-beef, causing high economical losses in the meat industry. Under this framework, the assessment of apoptosis onset in post mortem muscle was proposed as a novel approach to reveal biochemical characteristics in several Spanish bovine breeds (Asturiana de los Valles, Retinta and Rubia Gallega) managed under different production systems (intensive versus semi-extensive) and transport/lairage conditions (mixing versus not mixing with unfamiliar animals). To do so, the activities of initiator caspase 9 and executioner caspases 3/7 were determined in Longissimus thoracis et lumborum muscle at three early post mortem times (2, 8, and 24 h). RESULTS: Breed effect and transport/lairage conditions were the most relevant factors that influenced both caspase activities over post mortem time, showing Rubia Gallega breed a completely different behavior compared to Asturiana de los Valles and Retinta breeds. Moreover, it is postulated that apoptosis cascade is initiated via the activation of caspase 9 under hypoxic or metabolic stress followed by the activation of executioner caspases 3/7. CONCLUSIONS: Assessment of apoptosis on post mortem muscle can be a novel approach to study the influence of animal handling on muscle metabolism and post mortem cell death and its consequences on meat quality traits. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Suppression of tumorigenicity 2 after exercise: a systematic review.

Exercise is a pivotal physiological activity, associated with benefits. Whilst the importance of physical activity is consensual along different steps of the cardiovascular (CV) continuum, there has been interest in assessing the CV adaptations to vigorous exercise. Indeed, exercise can be associated with increases in cardiac biomarkers, though the scope of this observation remains elusive. Interleukin 1 receptor related protein, Suppression of tumorigenicity 2 (ST2) is a biomarker related to the pathophysiology of fibrosis, having shown promise in the study of heart failure. Knowledge of ST2 kinetics could improve understanding of the mechanistic pathways related to CV adaptations to exercise. To assess the current state-of-the-art concerning ST2 levels after exercise in healthy individuals. A systematic review was carried out on three databases (Pubmed, ISI Web of Science and Scopus), up to October 2020, using the queries "ST2" or "ST-2" + "exercise" or "running". A total of six studies were included in the review, encompassing 349 subjects (73% male gender) in which ST2 was assessed. Most studies reported increases in ST2 levels after exercise. Three studies, encompassing a total of 219 individuals, described a cut-off level of 35 ng/dL for ST2. In these, 92.7% of subjects had ST2 levels above this cut-off after exercise (running in all studies). Most studies report increased levels of ST2 after exercise, with an important number of individuals exceeding the 35 ng/dL threshold. Given the small number of individuals represented and the lack of imaging data and long-term follow-up, further prospective larger studies should target this.

Differential Impact of a Cardiac Rehabilitation Program on Functional Parameters in Elderly versus Non-Elderly Myocardial Infarction Survivors.

BACKGROUND: Exercise-based cardiac rehabilitation (EBCR) plays a pivotal role in the management of acute myocardial infarction (AMI). Studies have shown that older individuals have a worse prognosis after an AMI, attesting to the importance of risk reduction strategies. We aimed at assessing the impact of age (patients dichotomized as ≥65 years old or <65 years old) on the functional benefits of an EBCR program among AMI survivors. DESIGN: Observational, retrospective cohort study. PARTICIPANTS: All patients admitted due to an AMI who completed a phase II EBCR program after discharge, between November 2012 and April 2017. INTERVENTION: EBCR program. MEASUREMENTS: Functional parameters were assessed by a symptom-limited cardiopulmonary exercise test. RESULTS: A total of 379 patients were included (30% aged ≥65 years). After the EBCR program, peak oxygen uptake (pVO2) and exercise duration increased significantly. Patients aged ≥65 years presented with more comorbidities and a lower functional capacity. Those aged ≥65 years presented significantly smaller improvements in pVO2 (0.79 ± 2.61 vs. 1.60 ± 3.11 mL/kg/min, p = 0.016) and exercise duration [75 (59-120) vs. 120 s (60-180), p = 0.002]. This was maintained after adjusting for several potential confounders. CONCLUSION: Older patients have a worse functional capacity than their younger counterparts. Still, a contemporary EBCR program was associated with significant functional improvements among those aged ≥65 years. The smaller improvements even after adjustments for potential confounders suggest that physiological differences may contribute to this finding. These results highlight the relevance of EBCR among this higher-risk subgroup.

[Profiles of loneliness and social isolation in urban population]. / Perfiles de soledad y aislamiento social en población urbana.

OBJECTIVE: To assess the prevalence of loneliness and social isolation in a population over 65 cared by a urban primary health team and to identify its main characteristics. DESIGN: Cross-sectional descriptive study by a telephone survey. SETTING: Basic health area of Barcelona. PARTICIPANTS: Random sample of assigned population of 65 or more years old. MAIN MEASUREMENTS: UCLA Loneliness Scale and Lubben Social Network Scale. RESULTS: 278 persons were interviewed (61,36% response rate), 172 women and 106 men, with an average age of 76,7 ± 7,9 years. A higher proportion of factors related to loneliness were identified in non-respondents. Loneliness was closely correlated to social r = 0,736. Moderate loneliness, with a prevalence of 16,54%, was associated to walking difficulties (OR 3,09, 95%, IC 1,03-9,29), cognitive impairment (OR 3,97, 95% IC 1,19-13,27) and architectural barriers (OR 5.29, 95% IC 2.12-13,23), although severe loneliness, with a prevalence of 18,71% was only associated to living together with less people (OR 0.61, 95% IC 0.40-0.93). Social isolation, with a prevalence of 38,85% was associated to aging (OR 1,06, 95% IC 1,02-1,10) and to the belief of having health problems (OR 4,35, 95% IC 1,11-16,99). CONCLUSIONS: Loneliness and social isolation are of high prevalence. The telephone survey underestimates its prevalence. There are 2profiles of loneliness, one with moderate associated to the socialisation difficulties related to aging and another severe not related to health or to barriers that only can be identified by surveys or clinical interview. Interventions must be targeted to each of these profiles.

A Nature-Inspired Design Yields a New Class of Steroids Against Trypanosomatids.

Chagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as public health problems by the World Health Organization. These diseases affect millions of people around the world however, efficient and low-cost treatments are not available. Different steroid molecules with antimicrobial and antiparasitic activity were isolated from diverse organisms (ticks, plants, fungi). These molecules have complex structures that make de novo synthesis extremely difficult. In this work, we designed new and simpler compounds with antiparasitic potential inspired in natural steroids and synthesized a series of nineteen steroidal arylideneketones and thiazolidenehydrazines. We explored their biological activity against Leishmania infantum, Leishmania amazonensis, and Trypanosoma cruzi in vitro and in vivo. We also assayed their genotoxicity and acute toxicity in vitro and in mice. The best compound, a steroidal thiosemicarbazone compound 8 (ID_1260) was active in vitro (IC50 200 nM) and in vivo (60% infection reduction at 50 mg/kg) in Leishmania and T. cruzi. It also has low toxicity in vitro and in vivo (LD50 >2000 mg/kg) and no genotoxic effects, being a promising compound for anti-trypanosomatid drug development.

Complete atrioventricular block during exercise: New insights from an old test.

Exercise stress testing can have a central role in the assessment of cardiovascular disease. Contemporary data, however, has highlighted the added value of imaging modalities over the exercise electrocardiogram in the investigation of coronary artery disease. Given the physiological changes associated with exercise and the possibility to address other parameters such as rhythm changes and the chronotropic response, exercise stress testing with continuous electrocardiographic monitoring can still have an important place in contemporary clinical practice. We report the case of a complete atrioventricular block associated with exercise and discuss the current role of exercise stress testing with continuous electrocardiographic monitoring in this entity.

Looking for combination of benznidazole and Trypanosoma cruzi-triosephosphate isomerase inhibitors for Chagas disease treatment.

BACKGROUND: The current chemotherapy for Chagas disease is based on monopharmacology with low efficacy and drug tolerance. Polypharmacology is one of the strategies to overcome these limitations. OBJECTIVES: Study the anti-Trypanosoma cruzi activity of associations of benznidazole (Bnz) with three new synthetic T. cruzi-triosephosphate isomerase inhibitors, 2, 3, and 4, in order to potentiate their actions. METHODS: The in vitro effect of the drug combinations were determined constructing the corresponding isobolograms. In vivo activities were assessed using an acute murine model of Chagas disease evaluating parasitaemias, mortalities and IgG anti-T. cruzi antibodies. FINDINGS: The effect of Bnz combined with each of these compounds, on the growth of epimastigotes, indicated an additive action or a synergic action, when combining it with 2 or 3, respectively, and an antagonic action when combining it with 4. In vivo studies, for the two chosen combinations, 2 or 3 plus one fifth equivalent of Bnz, showed that Bnz can also potentiate the in vivo therapeutic effects. For both combinations a decrease in the number of trypomastigote and lower levels of anti-T. cruzi IgG-antibodies were detected, as well clear protection against death. MAIN CONCLUSIONS: These results suggest the studied combinations could be used in the treatment of Chagas disease.

Chemoenzymatic synthesis of optically active phenolic 3,4-dihydropyridin-2-ones: a way to access enantioenriched 1,4-dihydropyridine and benzodiazepine derivatives.

A chemoenzymatic approach for the synthesis of optically active 4-(3-acetoxyphenyl)-5-(alkoxycarbonyl)-6-methyl-3,4-dihydropyridin-2-ones (3,4-DHP-2-ones) and their hydroxyphenyl derivatives has been developed, the key step being a Candida rugosa lipase (CRL)-catalyzed hydrolysis reaction. As a result, different optically active 3,4-DHP-2-ones have been prepared with very high enantiomeric excesses (ee = 94-99%) and good yields. The enantioenriched 3,4-DHP-2-ones have easily been converted into highly functionalized (R)- and (S)-1,4-dihydropyridines (1,4-DHPs) by means of a Vilsmeier-Haack reaction. Finally, the coupling of the 1,4-DHPs with benzene-1,2-diamine using TFA as an acid promoter provided us the corresponding optically active hybrid 1,5-benzodiazepine-1,4-dihydropyridine (BZD-DHP) derivatives. No racemization took place in these processes and all optically active compounds were obtained in excellent yields.

Induction of viral interference by IPNV-carrier cells on target cells: A cell co-culture study.

IPNV is a salmonid birnavirus that possesses the ability to establish asymptomatic persistent infections in a number of valuable fish species. The presence of IPNV may interfere with subsequent infection by other viruses. In the present study we show that an IPNV-carrier cell line (EPCIPNV) can induce an antiviral state in fresh EPC by co-cultivating both cell types in three different ways: a "droplet" culture system, a plastic chamber setup, and a transmembrane (Transwell®) system. All three cell co-culture methods were proven useful to study donor/target cell interaction. Naïve EPC cells grown in contact with EPCIPNV cells develop resistance to VHSV superinfection. The transmembrane system seems best suited to examine gene expression in donor and target cells separately. Our findings point to the conclusion that one or more soluble factors produced by the IPNV carrier culture induce the innate immune response within the target cells. This antiviral response is associated to the up-regulation of interferon (ifn) and mx gene expression in target EPC cells. To our knowledge this is the first article describing co-culture systems to study the interplay between virus-carrier cells and naive cells in fish.

Identification of a new amide-containing thiazole as a drug candidate for treatment of Chagas' disease.

Although the parasitic infection Chagas' disease was described over 100 years ago, even now there are not suitable drugs. The available drugs nifurtimox and benznidazole have limited efficacies and tolerances, with proven mutagenic effects. Attempting to find appropriate drugs to deal with this problem, here we report on the development and pharmacological characterization of new amide-containing thiazoles. In the present study, we evaluated the in vitro and in vivo effects of new candidates against Trypanosoma cruzi, the etiological agent of Chagas' disease. The lead amide-containing thiazole derivative had potent in vitro activity, an absence of both in vitro mutagenic and in vivo clastogenic effects, and excellent in vitro selectivity and in vivo tolerance. The compound suppressed parasitemia in mice, modifying the anti-T. cruzi antibodies like the reference drug, benznidazole, and displayed the lowest mortality among the tested drugs. The present evidence suggests that this compound is a promising anti-T. cruzi agent surpassing the lead optimization stage in drug development and leading to a candidate for preclinical study.

In-office study to evaluate the effects of a stabilized stannous fluoride dentifrice and an advanced manual toothbrush regimen on gingival health and plaque accumulation in Mexican adults.

PURPOSE: This office-based study was conducted to investigate the influence of an oral hygiene regimen, consisting of a stabilized stannous fluoride dentifrice and an advanced manual toothbrush, on gingival health and plaque accumulation in Mexican adults after 4 weeks of use. METHODS: This was an unsupervised, open-label, single-treatment, 4-week study conducted in Mexico. At Baseline, gingival health and plaque coverage were assessed for each participant by dentists using categorical scales. Participants were given 0.454% stabilized stannous fluoride dentifrice (Crest Pro-Salud Limpieza Profunda) and an advanced manual toothbrush with a CrissCross bristle design (Oral-B Pro-Salud 7 Beneficios), and were instructed to follow the manufacturer's usage instructions for the products, which state to brush after every meal, at least three times daily, or as indicated by their dentists. At the end of Week 4, plaque and gingivitis were reassessed using the same scales. RESULTS: 200 participants (age range 18 - 49 years) completed the 4-week study and were considered evaluable. The mean age of the study group was 29.9 ± 8.8 years, with 125 females and 75 males. After 4 weeks of product use, 172 participants (86%) showed noticeable improvement in their gingival health and 93% showed noticeable improvements in their overnight plaque coverage.

In vivo anti-Trypanosoma cruzi activity of hydro-ethanolic extract and isolated active principles from Aristeguietia glutinosa and mechanism of action studies.

The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Attempting to find alternative drugs isolated from Nature as agents against Trypanosoma cruzi has been our goal. Recently, we have demonstrated the in vitro anti-T. cruzi activities of two secondary metabolites isolated from the hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa (Lam.), (family Asteraceae). These active principles displayed poor hemolytic activity, low toxicity against murine macrophages, and absence of mutagenicity. Herein, proof of concept in vivo studies of the whole hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa and of the most active component isolated from the hydro-ethanolic extract, i.e., (+)-15-hydroxy-7-labden-17-al, was done in a murine acute model of Chagas disease. Both treatments caused a decrease in the animals' parasitemia. Metabolomic mechanism of action studies were done by 1H-NMR, both on the extract and on the active compounds, examining the effects of the metabolites both on membrane sterol biosynthesis and mitochondrial dehydrogenases, whereby we found that one of the metabolites inhibited the activity of the parasite mitochondrial dehydrogenases and the other inhibited the biosynthesis of parasite membrane sterols. The results are interesting in the context of popular use of plants for the treatment of Chagas disease.

Exploring the miRNAs Profile in Dark-Cutting Beef.

In an animal production system, different stressors may cause the depletion of muscle glycogen stores, resulting in an elevated pH at 24 h post mortem (pH24), which leads to cell metabolism alterations that affect the conversion of muscle into meat, causing meat quality defects, such as dark-cutting beef, also known as dark, firm, and dry (DFD) beef. This process may involve the alteration of small non-coding RNAs (miRNAs), which play critical regulatory roles in cellular processes. Here, we determined whether differential miRNA expression in the Longissimus thoracis et lumborum muscle from the Asturiana de los Valles breed at 24 h post mortem could serve as an early indicator of beef quality defects. Following total RNA extraction, complete miRNAome sequencing revealed 12 miRNAs that were significantly upregulated (p < 0.001) in DFD beef compared to the levels in CONTROL beef. These miRNAs are mainly involved in the cellular responses to redox imbalances and apoptosis. Among these, four miRNAs known to be related to oxidative stress (bta-miR-1246, bta-miR-2332, bta-miR-23b-5p, and bta-miR-2411-3p) were validated via quantitative RT-PCR. Some of their target proteins were also analyzed using Western blotting. High 70 kDa heat shock protein and low Caspase-9 expressions (p < 0.01) were found in DFD beef, suggesting the downregulation of apoptosis. These results suggest the importance of miRNAs in regulating stress in muscle cells during early post mortem, as differences in the abundance of some of these miRNAs are still observed at 24 h post mortem. These changes lead to an inadequate conversion of muscle into meat, resulting in meats with quality defects.

Atomically accurate de novo design of single-domain antibodies.

Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.

De novo designed proteins neutralize lethal snake venom toxins.

Snakebite envenoming remains a devastating and neglected tropical disease, claiming over 100,000 lives annually and causing severe complications and long-lasting disabilities for many more1,2. Three-finger toxins (3FTx) are highly toxic components of elapid snake venoms that can cause diverse pathologies, including severe tissue damage3 and inhibition of nicotinic acetylcholine receptors (nAChRs) resulting in life-threatening neurotoxicity4. Currently, the only available treatments for snakebite consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs5,6,7. Here, we use deep learning methods to de novo design proteins to bind short- and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtain protein designs with remarkable thermal stability, high binding affinity, and near-atomic level agreement with the computational models. The designed proteins effectively neutralize all three 3FTx sub-families in vitro and protect mice from a lethal neurotoxin challenge. Such potent, stable, and readily manufacturable toxin-neutralizing proteins could provide the basis for safer, cost-effective, and widely accessible next-generation antivenom therapeutics. Beyond snakebite, our computational design methodology should help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for development of therapies to neglected tropical diseases.

Sixty years of the Bruce protocol: reappraising the contemporary role of exercise stress testing with electrocardiographic monitoring.

The cardiovascular response to exercise has long been a focus of interest. Over a century ago, the first descriptions of electrocardiographic changes occurring during exercise highlighted the possible relevance of this dynamic assessment. In this background, the inception of the Bruce protocol circa 60 years ago allowed for a major leap in this field by providing a standardized framework with which to address this issue, by means of an integrated and structured methodology. Since then, exercise stress testing with electrocardiographic monitoring (ExECG) has become one of the most widely appraised tests in cardiovascular medicine. Notably, past few decades have been profoundly marked by substantial advances in the approach to cardiovascular disease, challenging prior notions concerning both its physiopathology and overall management. Among these, the ever-evolving presentations of cardiovascular disease coupled with the development and implementation of several novel diagnostic modalities (both invasive and noninvasive) has led to a shifting paradigm in the application of ExECG. This technique, however, has continuously shown to be of added value across various momentums of the cardiovascular continuum, as depicted in several contemporary guidelines. This review provides a pragmatical reflexion on the development of ExECG, presenting a comprehensive overview concerning the current role of this modality, its challenges, and its future perspectives.