Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA-protein coupling.

Funder: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research); doi: https://doi.org/10.13039/501100003246Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein-DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes

Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process

: Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

[Asociación entre conocimientos y actitudes preventivas sobre complicaciones crónicas en diabéticos de un policlínico peruano]

Introducción: Hay escasos estudios realizados para valorar la asociación entre el nivel de conocimientos sobre la diabetes mellitus tipo 2 y las actitudes que tienen los pacientes para mejorar el control de su enfermedad. Objetivo: Determinar la asociación entre el nivel de conocimientos y las actitudes preventivas sobre las complicaciones crónicas en pacientes con diabetes mellitus tipo 2. Métodos: Estudio transversal analítico en pacientes con diabetes tipo 2 de Chiclayo, Perú. Se indagó la asociación entre actitudes preventivas y nivel de conocimientos, además se exploró asociación con edad, sexo, nivel de instrucción, instrucción diabetológica previa, antecedentes familiares, hospitalizaciones previas, tiempo de enfermedad. Resultados: De 150 pacientes, el 60 % fueron mujeres. El 40,0 % presentó un nivel de conocimientos intermedio y un 84,7 % reportaron actitudes preventivas favorables. Se encontró que los pacientes con nivel de conocimiento adecuado tenían 43 % mayor frecuencia de presentar actitudes preventivas favorables (razón de prevalencia = 1,43). Los pacientes que tenían entre 7 a 15 años de enfermedad resultaron asociados positivamente a tener actitudes preventivas favorables (razón de prevalencia = 1,32). Conclusiones: Los pacientes diabéticos con conocimientos adecuados sobre su enfermedad tienen actitudes preventivas favorables frente a su padecimiento y sus complicaciones crónicas. Adicionalmente, los pacientes diagnosticados entre 7 - 15 años atrás tienen mejores actitudes preventivas

Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling

Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein–DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes.BN/Sander Tans La

Weak catch bonds make strong networks

Molecular catch bonds are ubiquitous in biology and essential for processes like leucocyte extravasion1 and cellular mechanosensing2. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this feature provides ‘strength on demand3’, thus enabling cells to increase rigidity under stress1,4,5,6. However, catch bonds are often weaker than slip bonds because they have cryptic binding sites that are usually buried7,8. Here we show that catch bonds render reconstituted cytoskeletal actin networks stronger than slip bonds, even though the individual bonds are weaker. Simulations show that slip bonds remain trapped in stress-free areas, whereas weak binding allows catch bonds to mitigate crack initiation by moving to high-tension areas. This ‘dissociation on demand’ explains how cells combine mechanical strength with the adaptability required for shape change, and is relevant to diseases where catch bonding is compromised7,9, including focal segmental glomerulosclerosis10 caused by the α-actinin-4 mutant studied here. We surmise that catch bonds are the key to create life-like materials

Six years of experience in percutaneous closure of interatrial septal defects

MARCO DE REFERENCIA: los defectos del tabique interauricular son anormalidades congénitas del tabique interauricular que comprenden la comunicación interauricular y el foramen oval permeable. OBJETIVO: analizar y evaluar los resultados del cierre percutáneo con dispositivo percutáneo de pacientes con defectos del tabique interauricular en el Hospital Universitario Santa Fe de Bogotá desde la introducción de esta técnica en 2005 hasta 2011. MATERIALES Y MÉTODOS: se realizó un estudio descriptivo ambispectivo. La población estuvo conformada por pacientes adultos, independiente de edad y género, a quienes se les realizó cierre percutáneo de cualquier defecto del septo interauricular desde la introducción de esta técnica en enero 1º. de 2005 hasta junio de 2011 en el servicio de hemodinamia del Hospital Universitario Fundación Santa Fe de Bogotá. RESULTADOS: durante el periodo se hicieron 53 procedimientos de corrección de defecto del tabique interauricular por vía percutánea, en los que se usó dispositivo Amplatzer en 94,3% de los casos. El 75% (27 pacientes) se trataron de manera ambulatoria, dándose de alta luego de cuatro horas de efectuado el procedimiento. El 29,8% fueron hombres y 70,2% mujeres, con edad promedio de 52,2 ± 15,1 años. 57,8% de los pacientes tuvo foramen oval permeable y de éstos 54,5% tenía aneurisma asociado; el porcentaje restante, 42%, fue intervenido por comunicación interauricular. CONCLUSIONES: la experiencia en la Fundación Santa Fe de Bogotá muestra un excelente resultado con muy baja tasa de complicaciones, mejorías clínicas en el seguimiento a largo plazo y gran seguridad, factores que permiten que este procedimiento se lleve a cabo de manera ambulatoria.CONTEXT: atrial septal defects are congenital atrial septal abnormalities that comprise the interatrial communication (IAC) and the patent foramen ovale (PFO). OBJECTIVE: to analyze and evaluate the results of percutaneous closure with device in patients with interatrial septal defects in the University Hospital Santa Fe de Bogotá since the introduction of this technique in 2005 to 2011. MATERIALS AND METHODS: descriptive ambispective study. The population consisted of adult patients, regardless of age and gender, who underwent percutaneous closure of any atrial septal defect since the introduction of this technique in January 1st. 2005 to June 2011 in the service of hemodynamics of the University Hospital Fundación Santa Fe de Bogotá. RESULTS: during this period, 53 procedures of percutaneous correction of the atrial septal defect were performed, using the Amplatzer device in 94.3% cases. 75% (27 patients) were treated on an outpatient basis, being discharged four hours after the performance of the procedure. 29.8% were men and 70.2% women with mean age 52.2 ± 15.1 years. 57.8% of patients had patent foramen ovale and of these, 54.5% had associated aneurysm. The remainder 42%, was operated for interatrial communication. CONCLUSIONS: the experience in the Fundación Santa Fe de Bogota shows excellent results with a very low complication rate and clinical improvements in the long-term follow-up, factors that allow that this procedure can be performed on an outpatient basis

Protein chain collapse modulation and folding stimulation by GroEL-ES

The collapse of polypeptides is thought important to protein folding, aggregation, intrinsic disorder, and phase separation. However, whether polypeptide collapse is modulated in cells to control protein states is unclear. Here, using integrated protein manipulation and imaging, we show that the chaperonin GroEL-ES can accelerate the folding of proteins by strengthening their collapse. GroEL induces contractile forces in substrate chains, which draws them into the cavity and triggers a general compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL and is aided by GroES binding to the cavity rim and by the amphiphilic C-terminal tails at the cavity bottom. Collapse modulation is distinct from other proposed GroEL-ES folding acceleration mechanisms, including steric confinement and misfold unfolding. Given the prevalence of collapse throughout the proteome, we conjecture that collapse modulation is more generally relevant within the protein quality control machinery. BN/Sander Tans La

New Developments in NMR

Regulating protein states is considered the core function of chaperones. However, despite their importance to all major cellular processes, the conformational changes that chaperones impart on polypeptide chains are difficult to study directly due to their heterogeneous, dynamic, and multi-step nature. Here, we review recent advances towards this aim using single-molecule manipulation methods, which are rapidly revealing new mechanisms of conformational control and helping to define a different perspective on the chaperone function