RESUMEN

Although, in randomized clinical trials, once-weekly subcutaneous semaglutide (OW s.c.) has demonstrated superior efficacy in comparison with placebo and active controls in terms of glycemic control and body weight reduction in patients with type 2 diabetes mellitus (T2DM), these results need to be confirmed in a real-world (RW) setting. An RW ambispective study (6 months retrospective and 6 months prospective) was conducted in 10 tertiary hospitals in Spain. We evaluated changes in HbA1c and body weight in patients with T2DM treated with semaglutide OW s.c. Additionally, we analyzed different subgroups of patients treated with semaglutide OW s.c. as an add-on to glucose-lowering therapy. A total of 752 patients with a mean age of 60.2 years, a mean HbA1c level of 8.5%, a mean body weight of 101.6 kg, and a mean T2DM duration of 10 years were included. At 12 months, compared with baseline, there was a mean difference of -2.1% in HbA1c levels (p < 0.001) and a mean difference of 9.2 kg in body weight (p < 0.001). Moreover, there were statistically significant differences (p < 0.001) between baseline and month 12 in both HbA1c and body weight in the four subgroups receiving semaglutide OW s.c. as an add-on to glucose-lowering therapy. Semaglutide OW s.c. was well tolerated, with gastrointestinal disorders being the most commonly reported side effects. In this RW study, 12 months of treatment with semaglutide OW s.c. in patients with T2DM was associated with significant and clinically relevant improvements in glycemic control and weight loss, regardless of the glucose-lowering therapy received, and the overall safety profile was positive.

Asunto(s)

RESUMEN

Targeted protein degradation (TPD) modulates protein function beyond inhibition of enzyme activity or protein-protein interactions. Most degraders function by proximity induction, and directly bridge an E3 ligase with the target to be degraded. However, many proteins might not be addressable via proximity-based degraders, and other challenges, such as resistance acquisition, exist. Here, we identified pseudo-natural products derived from (-)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs induce a unique conformational change and, thereby, boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2KLHDC3, which we identified to also mediate native IDO1 degradation. Therefore, iDegs supercharge the native proteolytic pathway of IDO1, rendering this mechanism of action distinct from traditional degrader approaches involving proteolysis-targeting chimeras (PROTACs) or molecular-glue degraders (MGDs). In contrast to clinically explored IDO1 inhibitors, iDegs reduce formation of kynurenine by both inhibition and induced degradation of the enzyme and should also modulate non-enzymatic functions of IDO1. This unique mechanism of action may open up new therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

RESUMEN

Leachables in pharmaceutical products may react with biomolecule active pharmaceutical ingredients (APIs), for example, monoclonal antibodies (mAb), peptides, and ribonucleic acids (RNA), potentially compromising product safety and efficacy or impacting quality attributes. This investigation explored a series of in silico models to screen extractables and leachables to assess their possible reactivity with biomolecules. These in silico models were applied to collections of known leachables to identify functional and structural chemical classes likely to be flagged by these in silico approaches. Flagged leachable functional classes included antimicrobials, colorants, and film-forming agents, whereas specific chemical classes included epoxides, acrylates, and quinones. In addition, a dataset of 22 leachables with experimental data indicating their interaction with insulin glargine was used to evaluate whether one or more in silico methods are fit-for-purpose as a preliminary screen for assessing this biomolecule reactivity. Analysis of the data showed that the sensitivity of an in silico screen using multiple methodologies was 80%-90% and the specificity was 58%-92%. A workflow supporting the use of in silico methods in this field is proposed based on both the results from this assessment and best practices in the field of computational modeling and quality risk management.

Asunto(s)

RESUMEN

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Šfrom the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

Asunto(s)

RESUMEN

Measuring the sociocultural productivity of heritage sites remains an ongoing issue for international organizations concerned with the conservation and promotion of traditional sites. The productivity of these locations is not only affected by tangible elements but also by intangible factors, such as the emotions generated by the experiences. For this purpose, 597 employees of hotels in these historical locations who had visited one of the 14 heritage sites in Spain assessed what role emotions play in this contribution. The methodology used was the application of structural equations. Several conclusions have been drawn utilizing the SmartPLS 4 software. The first is that the generation of positive emotions comes exclusively from cultural and historical dynamization and not from technological advances or an eagerness to learn. The second is that both the application of technological advances and cultural dynamization have a direct impact on productivity.

RESUMEN

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.

RESUMEN

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Asunto(s)

Asunto(s)

RESUMEN

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

Asunto(s)

RESUMEN

RESUMO Introdução: O ensino baseado em competências no âmbito das residências médicas tornou evidente o descompasso dos processos de avaliação tradicionais com os objetivos educacionais dos projetos pedagógicos alinhados às matrizes de competências de cada especialidade. A matriz de competência para o Programa de Residência em Medicina Intensiva (acesso direto em três anos) foi aprovada em 2021. O objetivo deste artigo é descrever o relato de experiência de um projeto de intervenção nos instrumentos de avaliação de desempenho dos residentes no Programa em Residência em Medicina Intensiva de um hospital público universitário em São Luís, no Maranhão. Relato de experiência: Após a organização do grupo de estudo e de trabalho para a intervenção, houve a escolha do objeto "ferramentas de avaliação de competências" e a seleção do Programa de Residência de Medicina Intensiva. Inicialmente, foi aplicado um questionário a todos os médicos preceptores e residentes, com atuação no cenário da unidade de terapia intensiva (UTI), com o objetivo de aferir as percepções deles acerca do instrumento avaliativo vigente, seguindo a pergunta norteadora: "A avaliação atual atende à concepção do programa traduzido pela matriz de competência da Comissão Nacional de Residência Médica?". Discussão: Embora a maioria dos preceptores e residentes tenha considerado que os métodos de avaliação atendiam à concepção do programa, havia pontos frágeis em relação ao feedback e à avaliação de desempenho dos residentes. Como intervenção, propusemos adaptação da ferramenta existente, adequando-a aos desempenhos previstos na matriz de competências da especialidade com formalização do feedback e introdução de avaliação de desempenho em cenário real utilizando o Miniexercício Clínico Avaliativo (Mini-Cex). Conclusão: Os limites entre a avaliação e a aprendizagem são tênues. Com base em indicadores sobre a percepção de preceptores e residentes de fragilidades na avaliação utilizada de longa data, foi proposta uma intervenção de modificação dos instrumentos avaliativos com o intuito de adequar/melhorar a avaliação de competências.

ABSTRACT Introduction: Competency-based teaching in medical residencies has evidenced the mismatch between traditional assessment processes and the educational objectives of pedagogical projects aligned with the competency matrices of each specialty. The competency matrix for the Intensive Care Medicine Residency Program (3-year direct admission) was approved in 2021. The objective of this article is to describe an experience report of an intervention project in the performance assessment instruments of residents attending the Intensive Care Medicine Residency Program at a university hospital in São Luís-Maranhão. Experience Report: After organizing the study and working group for the intervention, the object "skills assessment tools" was chosen and the Intensive Care Medicine Residency Program was selected. Initially, a questionnaire was applied to all preceptors and residents working in the intensive care unit (ICU) setting with the aim of evaluating their perceptions regarding the current assessment tool, following the guiding question: does the current assessment meet the conception of the program translated by the competency matrix of the National Medical Residency Commission? Discussion: Although the majority of the preceptors and residents considered that the evaluation methods met the Program design, there were weaknesses in relation to feedback and evaluation of the residents' performance. As an intervention, we proposed adapting the existing tool, making it adequate to the performance predicted in the specialty competency matrix with formalization of feedback and introduction of performance assessment in a real scenario using the Mini Clinical Assessment Exercise (Mini-CEX). Conclusion: The boundaries between assessment and learning are blurred. Based on indicators on the perception of preceptors and residents of weaknesses in the assessment used for a long time, an intervention was proposed to modify the assessment instruments with the aim of adapting/improving the assessment of skills.

RESUMEN

Whiplash-associated disorders (WAD) represent a multifactorial condition often accompanied by altered nociceptive processing and psychological factors. This systematic review on acute and chronic WAD aimed to investigate the relationship between quantitative sensory testing (QST) and psychological factors and quantify whether their trajectories over time follow a similar pattern to disability levels. Eight databases were searched until October 2022. When 2 prospective studies examined the same QST or psychological variable, data synthesis was performed with random-effects meta-analysis by pooling within-group standardized mean differences from baseline to 3-, 6-, and 12-month follow-ups. From 5,754 studies, 49 comprising 3,825 WAD participants were eligible for the review and 14 for the data synthesis. Altered nociceptive processing in acute and chronic WAD, alongside worse scores on psychological factors, were identified. However, correlations between QST and psychological factors were heterogeneous and inconsistent. Furthermore, disability levels, some QST measures, and psychological factors followed general positive improvement over time, although there were differences in magnitude and temporal changes. These results may indicate that altered psychological factors and increased local pain sensitivity could play an important role in both acute and chronic WAD, although this does not exclude the potential influence of factors not explored in this review. PERSPECTIVE: Acute WAD show improvements in levels of disability and psychological factors before significant improvements in nociceptive processing are evident. Facilitated nociceptive processing might not be as important as psychological factors in chronic WAD-related disability, which indicates that chronic and acute WAD should not be considered the same entity although there are similarities. Nonetheless, pressure pain thresholds in the neck might be the most appropriate measure to monitor WAD progression.

Asunto(s)

RESUMEN

This study presents a geo-spatial and economic framework to localize future bioenergy power plants combined with direct air capture (BEDAC). This framework is applied to two regions in the USA to assess the optimal use of forest biomass and in situ carbon sequestration under three specific short-term sequestration targets. Results show that there are many locations that have both the necessary biomass and geology required for storage. The Southeast has greater potential for forestry biomass due to both the rate of growth and forested areas, but the sequestration potential is mostly limited to a CO2 solution in saline aquifers. The Pacific Northwest has more sequestration potential than the Southeast given the location of managed forests and storage sites in carbonate mineralization in bedrock. The two combined regions have a total potential sequestration of 9.3 GtCO2 for the next 20 years that can be achieved under an implicit carbon value of $249/tCO2.

Asunto(s)

RESUMEN

Objectives: Semaglutide is a glucagon-like peptide 1 receptor agonist that improves glycemic control and achieves weight loss in type 2 diabetes (T2D) patients. Subcutaneous (s.c.) semaglutide at 1 mg once weekly (OW) is safe in T2D patients with chronic kidney disease (CKD). Whether or not CKD and its severity influence treatment response remains undetermined. Method: This is an observational, ambispective, multicenter, nationwide, real-world study designed to compare safety/efficacy of OW s.c. 1 mg semaglutide in T2D patients with or without CKD. The influence of CKD severity was also addressed. Patients were followed up for 12 months. Primary end-points were glycosylated hemoglobin (HbA1c), weight, and renal outcomes. Secondary end-points included insulin resistance, atherogenic and hepatic steatosis indexes, and changes in antihyperglycemic medications. Results: A total of 296 and 190 T2D patients without or with CKD, respectively, were recruited. Baseline CKD risk was moderate, high, or very high in 82, 53, and 45 patients, respectively. Treatment reduced HbA1c by 0.90%-1.20%. Relevant differences were seen neither between non-CKD and CKD patients nor among CKD subgroups. Notable weight losses were achieved in both non-CKD and CKD patients. The median reduction was higher in the former at 6 months (5.90 kg vs. 4.50 kg, P = 0.008) and at end of study (6.90 kg vs. 5.00 kg, P = 0.087). A trend toward slightly lower weight losses as CKD severity increased was observed. CKD markers improved across all CKD subgroups. Relevant differences were not observed for other variables, either between non-CKD and CKD patients, or among CKD subgroups. Safety concerns were not reported. Conclusion: The safety/efficacy of OW s.c. semaglutide to improve glycemic control and weight in T2D patients with CKD is not notably lower than that in T2D patients without renal failure. CKD severity barely influences treatment response. OW s.c. semaglutide can be useful to manage T2D patients with CKD in daily clinical practice.

Asunto(s)

RESUMEN

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O2 to H2O2. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsG3Ox oxidizes at 50,000-fold higher specificity (kcat/Km) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsG3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.

Asunto(s)

RESUMEN

In recent years geoparks, helped by governmental policies, have become tourist destinations especially among senior visitors. The paper aimed to analyse whether geoparks contribute to improving the health of tourists older than 65 years and what were their main motives to visit geoparks. The data were collected from 398 senior tourists who visited the Villuerca- Ibores-Jara Geopark (Spain) in 2023, presenting our results using SmartPLS version 4. The results showed that senior tourists are very interested in visiting this geopark for psychotherapeutic reasons, given its high environmental and geological interest. In addition, they consider geoparks as spaces where they can socialise, which is beneficial considering the isolation that many often experience during the year. These findings are highly relevant for public authorities to protect, maintain and promote geoparks among senior tourists.

RESUMEN

The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying molecular mechanisms by which TOR might regulate events after anaphase remain unknown. We show for the first time that one of the 2 TOR complexes in budding yeast, TORC1, blocks the separation of cells following cytokinesis by phosphorylation of a member of the NDR (nuclear Dbf2-related) protein-kinase family, the protein Cbk1. We observe that TORC1 alters the phosphorylation pattern of Cbk1 and we identify a residue within Cbk1 activation loop, T574, for which a phosphomimetic substitution makes Cbk1 catalytically inactive and, indeed, reproduces TORC1 control over cell separation. In addition, we identify the exocyst component Sec3 as a key substrate of Cbk1, since Sec3 activates the SNARE complex to promote membrane fusion. TORC1 activity ultimately compromises the interaction between Sec3 and a t-SNARE component. Our data indicate that TORC1 negatively regulates cell separation in budding yeast by participating in Cbk1 phosphorylation, which in turn controls the fusion of secretory vesicles transporting hydrolase at the site of division.

Asunto(s)

RESUMEN

INTRODUCTION: This study explored the ability of plasma amyloid beta (Aß)42/Aß40 to identify brain amyloid deposition in cognitively unimpaired (CU) individuals. METHODS: Plasma Aß was quantified with an antibody-free high-performance liquid chromatography tandem mass spectrometry method from Araclon Biotech (ABtest-MS) in a subset of 731 CU individuals from the screening visit of the Anti-Amyloid Treatment in Asymptomatic Alzheimer's (A4) Study, to assess associations of Aß42/Aß40 with Aß positron emission tomography (PET). RESULTS: A model including Aß42/Aß40, age, apolipoprotein E ε4, and recruitment site identified Aß PET status with an area under the curve of 0.88 and an overall accuracy of 81%. A plasma-based pre-screening step could save up to 42% of the total number of Aß PET scans. DISCUSSION: ABtest-MS accurately identified brain amyloid deposition in a population of CU individuals, supporting its implementation in AD secondary prevention trials to reduce recruitment time and costs. Although a certain degree of heterogeneity is inherent to large and multicentric trials, ABtest-MS could be more robust to pre-analytical bias compared to other immunoprecipitation mass spectrometry methods. HIGHLIGHTS: Plasma amyloid beta (Aß)42/Aß40 accurately identified brain Aß deposition in cognitively unimpaired individuals from the Anti-Amyloid Treatment in Asymptomatic Alzheimer's (A4) Study.The inclusion of the recruitment site in the predictive models has a non-negligible effect.A plasma biomarker-based model could reduce recruitment costs in Alzheimer's disease secondary prevention trials.Antibody-free liquid chromatography mass spectrometry methods may be more robust to pre-analytical variability than other platforms.

RESUMEN

In recent years, simulations have been used to great advantage to understand the structural and dynamic aspects of distinct enzyme immobilization strategies, as experimental techniques have limitations in establishing their impact at the molecular level. In this review, we discuss how molecular dynamic simulations have been employed to characterize the surface phenomenon in the enzyme immobilization procedure, in an attempt to decipher its impact on the enzyme features, such as activity and stability. In particular, computational studies on the immobilization of enzymes using i) nanoparticles, ii) self-assembled monolayers, iii) graphene and carbon nanotubes, and iv) other surfaces are covered. Importantly, this thorough literature survey reveals that, while simulations have been primarily performed to rationalize the molecular aspects of the immobilization event, their use to predict adequate protocols that can control its impact on the enzyme properties is, up to date, mostly missing.

RESUMEN

BACKGROUND: The aim of this study is to investigate the use of once-weekly semaglutide in a real population of patients with type 2 diabetes mellitus (T2DM) over 70 years in two Spanish hospitals. METHODS: An observational, retrospective, and multicenter clinical study was designed. It included 60 patients with T2DM, with a mean age of 76.5 years, 63.3% women, and a mean of 15.5 years of evolution of T2DM, all managed in the outpatient clinical setting. The primary endpoint was the change in HbA1c from baseline to the end of the study. The secondary endpoints included changes in body weight and the proportion of patients achieving HbA1c <7.0% and body weight loss >5%. RESULTS: After 12 months of follow-up, the reductions in HbA1c were -0.61±0.9% (P<0.0001) in the total cohort. Body weight reductions were -8.2±5.3 kg (P<0.0001). Overall, 67% reached the objective of an HbA1c level of <7%, and 73% achieved a weight loss of ≥5%. CONCLUSIONS: In routine clinical practice in Spain, the use of semaglutide once a week was associated with statistically significant and clinically relevant improvements in HbA1c and body weight in adults aged over 70 years with T2DM, without notable adverse effects, which supports real-world use.