[Community reorientation of primary care in a health area: ROCAP project]. / Reorientación comunitaria de la atención primaria en un área de salud: proyecto ROCAP.

AIM: Evaluate the effectiveness of an intervention to incorporate group and community activities on a sustained basis in all the Basic Health Zones (ZBS) of a health area. DESIGN: During January and February 2019, two members of the research team traveled to each ZBS to interview the medical and nursing coordinators who had previously received an ad hoc initial assessment questionnaire (QAI) by email. PLACE: The scope is the 11 ZBS of a health area. PARTICIPANTS: The study population is the ZBS and the respective teams represented by the medical and nursing coordinations. INTERVENTIONS: Promote a community health commission, carry out training actions, record activities in clinical history and incorporate management objectives. MAIN MEASUREMENTS: Quantitative and qualitative analysis was carried out pre and post after the first year of intervention. RESULTS: In the pre-evaluation: 6 primary care teams (EAP) reported having group activities, 4 were participating in local action projects, 4 had a professional referent for community activities, 3 participated in projects with populations in vulnerable situations and 4 stated have specific meetings on community health. After the intervention: 11 EAPs had group activities, 8 had a reference professional, 6 were participating in local action projects, 4 collaborated in projects with vulnerable populations and 5 held meetings on community health. CONCLUSIONS: The intervention proved effective after its first year of implementation, since all the EAPs carried out group activities and collaborated with the local councils in the area; the majority had leading professionals in community care and, to a lesser extent, participation in local action projects and in vulnerable populations increased.

Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions.

The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

Calidad de atención en el manejo de enfermedad cerebrovascular isquémica aguda posterior a la iniciativa angels / Acute ischemic cerebrovascular disease management quality of care following the adoption of the angels initiative

Introducción: el ataque cerebrovascular (ACV) es común a nivel mundial, una de cada cuatro personas puede presentarlo a lo largo de la vida. Constituye la segunda causa de muerte y la tercera principal de discapacidad. Es importante la atención integral para lograr un impacto en la calidad de vida. Objetivo: determinar la calidad de atención en el manejo del ACV isquémico agudo de los pacientes que consultaron al servicio de neurología en los Hospitales de San José e Infantil Universitario de San José, Bogotá DC, entre enero 1/2019 y enero 1/2020. Metodología: estudio descriptivo de corte transversal. El criterio de inclusión fue pacientes mayores de 18 años con diagnóstico de ACV isquémico. La información se recolectó de las historias clínicas, se empleó estadística descriptiva para analizar los datos. Resultados: se incluyeron 411 pacientes, 88,8% sin alteración del estado de conciencia, 26,4% ingresaron antes de las 4,5 horas de ventana para trombólisis, se realizaron procedimientos de recanalización endovenosa a 11,4%. El tiempo puerta aguja tuvo una mediana de 37,2 minutos comparado con la mediana nacional de tiempo que fue 56,5 min según lo registrado en la plataforma ResQ. El 72% recibió terapia antiagregante y estatina 88.8%. Discusión y conclusiones: al identificar los síntomas es importante ser estrictos en el tiempo de atención y la implementación de plataformas para óptimos planes de atención. Se requieren campañas masivas de educación, así como que planes de mejora institucionales.

Introduction: cerebrovascular attack (CVA) is common worldwide. One in four people may have a stroke during their lifetime. It is the second leading cause of death and the third leading cause of disability. Thus, it is important to provide integrated care to achieve an impact on quality of life. Objective: to determine ischemic CVA management quality of care among patients seen at the neurology service of the San José and Infantil Universitario de San José hospitals in Bogotá DC, between January 1/2019 and January 1/2020. Methodology: a descriptive, cross-sectional study. The inclusion criteria included patients over 18 years of age diagnosed with ischemic CVA. Information was collected from clinical records. Data was analyzed using descriptive statistics. Results: 411 patients were included; 88.8% had an altered state of consciousness, 26.4% were admitted within the 4.5-hour window for thrombolysis; 11.4% underwent intravenous reperfusion procedures. Door-to-needle time: median was 37.2 minutes versus the national media of 56.5 min according to the ResQ records platform; 72% received anti-platelet therapy and 88.8% received statins. Discussion and conclusions: the establishment of a strict time to care and the implementation of platforms to improve care plans, is essential. Massive education campaigns are required, as well as, institutional improvement plans.

Síndrome de apnea del sueño en población colombiana con epilepsia empleando la escala de apnea del sueño del cuestionario de trastornos del sueño / Sleep apnea syndrome in a Colombian population with epilepsy using the Sleep Apnea Scale of the Sleep Disorders Questionnaire

Introducción: La epilepsia es un desorden caracterizado por la predisposición a generar crisis epilépticas, mientras que el síndrome de apnea del sueño (SAOS) ha sido reconocido como un desorden crónico de colapso intermitente de la vía aérea que genera hipoxia recurrente. En este trabajo se aplicó la escala de trastornos del sueño (Sleep Apnea Scale of the Sleep Disorders Questionnaire SA-SDQ), previamente validada en inglés para pacientes con epilepsia, a fin de determinar su capacidad para detectar apnea de sueño en nuestra población. Materiales y métodos: En una primera etapa se realizó la adaptación transcultural de la escala SA-SDQ en castellano, provista por los autores, al español colombiano. Luego se recopiló la información de los pacientes en quienes se realizó polisomnografía entre mayo y agosto del 2022 y se determinó el valor de corte para diagnosticar SAOS con la escala SA-SDQ. Resultados: Cuarenta pacientes pudieron realizarse la polisomnografía, de los cuales 30 (75 %) tuvieron índices de apnea-hipopnea superiores a 5, lo que indica SAOS. El área bajo la curva fue 0,790 y la puntuación SA-SDQ de 21 proporcionó una sensibilidad del 73,3 % (IC 53,83-87,02 %) y una especificidad del 80 % (IC 44,2-96,5 %). La consistencia interna fue aceptable (α = 0,713). Conclusiones: La escala SA-SDQ es un instrumento útil para tamizar SAOS en la población colombiana que padece epilepsia. Nuestros resultados indican que los puntos de corte sugeridos anteriormente (2936 para hombres y 26-32 para mujeres) pueden ser demasiado altos para nuestra población. Sugerimos un punto de corte de 21 para ambos.

Introduction: Epilepsy is a disorder characterized by a predisposition to have epileptic seizures, while sleep apnea syndrome (OSAS) has been recognized as a chronic disorder of intermittent collapse of the airway that generates recurrent hypoxia. In this work, the sleep disorders scale (SA-SDQ) previously validated in English for patients with epilepsy was applied to determine its ability to detect sleep apnea in our population. Materials and methods: In the first stage, the cross-cultural adaptation of the SA-SDQ scale in Spanish provided by the authors was carried out into Colombian Spanish. then the information of the patients in whom polysomnography was performed between May and August 2022 was collected and the cut-off value was determined to diagnose OSAS with the SA-SDQ scale. Results: 40 patients were able to undergo polysomnography, of which 30 (75 %) had apnea-hypopnea indices greater than five, indicating OSAS. The area under the curve was 0.790 and the SA-SDQ score of 21 provided a sensitivity of 73.3 % (CI 53.83-87.02 %) and a specificity of 80 % (CI 44.2-96, 5 %). The internal consistency was acceptable (α = 0.713). Conclusions: The SA-SDQ scale is a useful instrument for screening OSAS in the Colombian population suffering from epilepsy. Our results indicate that the previously suggested cut-off points (29-36 for men and 26-32 for women) may be too high in our population. We suggest a cutoff of 21 for both.

A PAK1 Mutational Hotspot Within the Regulatory CRIPaK Domain is Associated With Severe Neurodevelopmental Disorders in Children.

BACKGROUND: P-21-activated kinases (PAKs) are protein serine/threonine kinases, part of the RAS/mitogen-activated protein kinase pathway. PAK1 is highly expressed in the central nervous system and crucially involved in neuronal migration and brain developmental processes. Recently, de novo heterozygous missense variants in PAK1 have been identified as an ultrarare cause of pediatric neurodevelopmental disorders. METHODS: We report a series of children affected with postnatal macrocephaly, neurodevelopmental impairment, and drug-resistant epilepsy. Repeated electroencephalographic (EEG) and video-EEG evaluations were performed over a two- to 10-year period during follow-up to delineate electroclinical histories. Genetic sequencing studies and computational evaluation of the identified variants were performed in our patient cohort. RESULTS: We identified by whole-exome sequencing three novel de novo variants in PAK1 (NM_001128620: c.427A>G, p.Met143Val; c.428T>C, p.Met143Thr; c.428T>A, p.Met143Lys) as the underlying cause of the disease in our families. The three variants affected the same highly conserved Met143 residue within the cysteine-rich inhibitor of PAK1 (CRIPaK) domain, which was identified before as a PAK1 inhibitor target. Computational studies suggested a defective autoinhibition presumably due to impaired PAK1 autoregulation as a result of the recurrent substitution. CONCLUSIONS: We delineated the electroclinical phenotypes of PAK1-related neurological disorders and highlight a novel mutational hotspot that may involve defective autoinhibition of the PAK1 protein. The three novel variants affecting the same hotspot residue within the CRIPaK domain highlight potentially impaired PAK1-CRIPaK interaction as a novel disease mechanism. These findings shed light on possible future treatments targeted at the CRIPaK domain, to modulate PAK1 activity and function.

Description of conformational ensembles of disordered proteins by residue-local probabilities.

The study of proteins with intrinsically disordered regions (IDRs) has emerged as an active field of research due to their intriguing nature. Although IDRs lack a well-defined folded structure, they play important functional roles in cells, following biological mechanisms different from those of the traditional structured proteins. Consequently, it has been necessary to re-design experimental and theoretical methods in order to face the challenges introduced by the dynamic nature of IDRs. In this work, we present an accurate and cost-effective method to study the conformational dynamics of IDRs based on the use of residue-local probabilistic expressions that characterize the conformational ensembles obtained from finite-temperature molecular dynamics (MD) simulations. It is shown that the good performance and the high convergence rates achieved with our method are independent of the IDR lengths, since the method takes advantage of the major influence of the identity and conformation of the nearest residue neighbors on the amino-acid conformational preferences to evaluate the IDR conformational ensembles. This allows us to characterize the conformational space of IDRs using a reduced number of probabilities which can be obtained from comparatively short MD simulations or experimental databases. To exemplify the usefulness of our approach, we present an application to directly detect Molecular Recognition Features (MoRFs) in an IDR domain of the protein p53, and to follow the time evolution of the thermodynamic magnitudes of this system during its exploration of the conformational space.

Replica-exchange optimization of antibody fragments.

In the framework of the rational design of macromolecules capable of binding to a specific target for biosensing applications, we here further develop an evolutionary protocol designed to optimize the binding affinity of protein binders. In particular we focus on the optimization of the binding portion of small antibody fragments known as nanobodies (or VHH) and choose the hen egg white lysozyme (HEWL) as our target. By implementing a replica exchange scheme for this optimization, we show that an initial hit is not needed and similar solutions can be found by either optimizing an already known anti-HEWL VHH or a randomly selected binder (here a VHH selective towards another macromolecule). While we believe that exhaustive searches of the mutation space are most appropriate when only few key residues have to be optimized, in case a lead binder is not available the proposed evolutionary algorithm should be instead the method of choice.

Current state of telemedicine in the field of Hepatology in Spain: challenges, threats and next steps to follow for an intelligent digital transformation.

Medicine and technology are constantly evolving. The COVID-19 pandemic has accelerated the development of digitalization in the health sector and specifically of telemedicine. Through a structured bibliographic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) methodology, in this study, the concepts related to telemedicine, its application and the legal regulatory context are defined. With this information, some recommendations and codes of good practice are proposed for their effective implementation in the field of Hepatology.

Antibody Affinity Maturation Using Computational Methods: From an Initial Hit to Small-Scale Expression of Optimized Binders.

Nanobodies (VHHs) are engineered fragments of the camelid single-chain immunoglobulins. The VHH domain contains the highly variable segments responsible for antigen recognition. VHHs can be easily produced as recombinant proteins. Their small size is a good advantage for in silico approaches. Computer methods represent a valuable strategy for the optimization and improvement of their binding affinity. They also allow for epitope selection offering the possibility to design new VHHs for regions of a target protein that are not naturally immunogenic. Here we present an in silico mutagenic protocol developed to improve the binding affinity of nanobodies together with the first step of their in vitro production. The method, already proven successful in improving the low Kd of a nanobody hit obtained by panning, can be employed for the ex novo design of antibody fragments against selected protein target epitopes.

A lipid transfer protein ensures nematode cuticular impermeability.

The cuticle of C. elegans is impermeable to chemicals, toxins, and pathogens. However, increased permeability is a desirable phenotype because it facilitates chemical uptake. Surface lipids contribute to the permeability barrier. Here, we identify the lipid transfer protein GMAP-1 as a critical element setting the permeability of the C. elegans cuticle. A gmap-1 deletion mutant increases cuticular permeability to sodium azide, levamisole, Hoechst, and DiI. Expressing GMAP-1 in the hypodermis or transiently in the adults is sufficient to rescue this gmap-1 permeability phenotype. GMAP-1 protein is secreted from the hypodermis to the aqueous fluid filling the space between collagen fibers of the cuticle. In vitro, GMAP-1 protein binds phosphatidylserine and phosphatidylcholine while in vivo, GMAP-1 sets the surface lipid composition and organization. Altogether, our results suggest GMAP-1 secreted by hypodermis shuttles lipids to the surface to form the permeability barrier of C. elegans.

Antibody-Antigen Binding Interface Analysis in the Big Data Era.

Antibodies have become the Swiss Army tool for molecular biology and nanotechnology. Their outstanding ability to specifically recognise molecular antigens allows their use in many different applications from medicine to the industry. Moreover, the improvement of conventional structural biology techniques (e.g., X-ray, NMR) as well as the emergence of new ones (e.g., Cryo-EM), have permitted in the last years a notable increase of resolved antibody-antigen structures. This offers a unique opportunity to perform an exhaustive structural analysis of antibody-antigen interfaces by employing the large amount of data available nowadays. To leverage this factor, different geometric as well as chemical descriptors were evaluated to perform a comprehensive characterization.

A homozygous MED11 C-terminal variant causes a lethal neurodegenerative disease.

PURPOSE: The mediator (MED) multisubunit-complex modulates the activity of the transcriptional machinery, and genetic defects in different MED subunits (17, 20, 27) have been implicated in neurologic diseases. In this study, we identified a recurrent homozygous variant in MED11 (c.325C>T; p.Arg109Ter) in 7 affected individuals from 5 unrelated families. METHODS: To investigate the genetic cause of the disease, exome or genome sequencing were performed in 5 unrelated families identified via different research networks and Matchmaker Exchange. Deep clinical and brain imaging evaluations were performed by clinical pediatric neurologists and neuroradiologists. The functional effect of the candidate variant on both MED11 RNA and protein was assessed using reverse transcriptase polymerase chain reaction and western blotting using fibroblast cell lines derived from 1 affected individual and controls and through computational approaches. Knockouts in zebrafish were generated using clustered regularly interspaced short palindromic repeats/Cas9. RESULTS: The disease was characterized by microcephaly, profound neurodevelopmental impairment, exaggerated startle response, myoclonic seizures, progressive widespread neurodegeneration, and premature death. Functional studies on patient-derived fibroblasts did not show a loss of protein function but rather disruption of the C-terminal of MED11, likely impairing binding to other MED subunits. A zebrafish knockout model recapitulates key clinical phenotypes. CONCLUSION: Loss of the C-terminal of MED subunit 11 may affect its binding efficiency to other MED subunits, thus implicating the MED-complex stability in brain development and neurodegeneration.

Genotype-phenotype correlations and disease mechanisms in PEX13-related Zellweger spectrum disorders.

BACKGROUND: Pathogenic variants in PEX-genes can affect peroxisome assembly and function and cause Zellweger spectrum disorders (ZSDs), characterized by variable phenotypes in terms of disease severity, age of onset and clinical presentations. So far, defects in at least 15 PEX-genes have been implicated in Mendelian diseases, but in some of the ultra-rare ZSD subtypes genotype-phenotype correlations and disease mechanisms remain elusive. METHODS: We report five families carrying biallelic variants in PEX13. The identified variants were initially evaluated by using a combination of computational approaches. Immunofluorescence and complementation studies on patient-derived fibroblasts were performed in two patients to investigate the cellular impact of the identified mutations. RESULTS: Three out of five families carried a recurrent p.Arg294Trp non-synonymous variant. Individuals affected with PEX13-related ZSD presented heterogeneous clinical features, including hypotonia, developmental regression, hearing/vision impairment, progressive spasticity and brain leukodystrophy. Computational predictions highlighted the involvement of the Arg294 residue in PEX13 homodimerization, and the analysis of blind docking predicted that the p.Arg294Trp variant alters the formation of dimers, impairing the stability of the PEX13/PEX14 translocation module. Studies on muscle tissues and patient-derived fibroblasts revealed biochemical alterations of mitochondrial function and identified mislocalized mitochondria and a reduced number of peroxisomes with abnormal PEX13 concentration. CONCLUSIONS: This study expands the phenotypic and mutational spectrum of PEX13-related ZSDs and also highlight a variety of disease mechanisms contributing to PEX13-related clinical phenotypes, including the emerging contribution of secondary mitochondrial dysfunction to the pathophysiology of ZSDs.

Computational Evolution Protocol for Peptide Design.

Computational peptide design is useful for therapeutics, diagnostics, and vaccine development. To select the most promising peptide candidates, the key is describing accurately the peptide-target interactions at the molecular level. We here review a computational peptide design protocol whose key feature is the use of all-atom explicit solvent molecular dynamics for describing the different peptide-target complexes explored during the optimization. We describe the milestones behind the development of this protocol, which is now implemented in an open-source code called PARCE. We provide a basic tutorial to run the code for an antibody fragment design example. Finally, we describe three additional applications of the method to design peptides for different targets, illustrating the broad scope of the proposed approach.

Enhanced Molecular Dynamics Method to Efficiently Increase the Discrimination Capability of Computational Protein-Protein Docking.

Protein-protein docking typically consists of the generation of putative binding conformations, which are subsequently ranked by fast heuristic scoring functions. The simplicity of these functions allows for computational efficiency but has severe repercussions on their discrimination capabilities. In this work, we show the effectiveness of suitable descriptors calculated along short scaled molecular dynamics runs in recognizing the nearest-native bound conformation among a set of putative structures generated by the HADDOCK tool for eight protein-protein systems.

Insights on peptide topology in the computational design of protein ligands: the example of lysozyme binding peptides.

Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for.

Parkinson genetico: Reporte de un caso clínico y revisión de la literatura / Genetic Parkinson disease: a case report and literature review

RESUMEN El parkinsonismo constituye un conjunto de signos y síntomas clínicos caracterizados por bradicinesia y temblor en reposo o rigidez, cuya causa más frecuente es la enfermedad de Parkinson (EP). La gran mayoría de los casos de EP son esporádicos, sin embargo, existe una minoría en la cual la etiología se debe a una mutación heredada, ya sea autosómica dominante (AD), autosómica recesiva (AR) o herencia ligada al X. La identificación de estas causas heredables es importante para una adecuada consejería genética y tratamiento. Se presenta el caso de un paciente con EP de inicio temprano en el que se identificó una mutación AD en el gen GIGYF2 o PARK11, asociado a una breve revisión de la literatura

SUMMARY Parkinsonism constitutes a set of clinical signs and symptoms characterized by bradykinesia and tremor at rest and / or rigidity. The main etiology is Parkinson's disease (PD), but there are other causes such as atypical parkinsonism. The vast majority of PD cases are sporadic, however, there is a minority where the etiology is due to an inherited mutation, either autosomal dominant (AD), autosomal recessive (RA), or X-linked inheritance. Identifying these heritable causes is important for proper genetic counseling and treatment. We present the case of a patient with early-onset PD where an AD mutation in the GIGYF2 gene (PARK11) was identified. We subsequently present a brief review of the literature.

[Adequacy of the safety of metamizole and agranulocytosis]. / Adecuación de la seguridad del metamizol y agranulocitosis.

OBJECTIVE: To analyze whether the drug safety update issued by the Spanish Agency of Medicines and Healthcare Products (AEMPS), dated October 30, 2018, on agranulocytosis and metamizole contains accurate and necessary information to protect patients from the presentation of this adverse reaction (AR) and if the official documentation of medicines containing metamizole for doctors, pharmacists and the general population conforms to the guidelines of the AEMPS to reduce this risk. SETTING AND PARTICIPANTS: Drug safety update, bibliographic search, information at the European Medicines Agency on metamizole drugs marketed in Spain, technical datasheets, leaflets, Bot PLUS Health Information Database and Catalog of Pharmaceutical Specialties. Notification of 4cases of agranulocytosis due to metamizole after the drug safety update was issued. MAIN INTERVENTIONS AND MEASUREMENTS: Comparison of the key points of the drug safety update and official documents on metamizole with the bibliography. Description of the 4cases of agranulocytosis due to metamizole and application of the causality and severity algorithm. RESULTS: The drug safety update contains omissions and contradiction in respect to the bibliography and the actual use of metamizole in healthcare practice. The official documents show a lack of updating, unapproved indications and doses higher than those recommended. The drug safety update has not stopped the presentation of cases of agranulocytosis due to metamizole. CONCLUSIONS: The AEMPS drug safety update can be improved and it is necessary to update the official information documents on metamizole for health professionals and patients in order to decrease the risk of agranulocytosis.

Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region.

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains.

Computational Evolution of Beta-2-Microglobulin Binding Peptides for Nanopatterned Surface Sensors.

The bottom-up design of smart nanodevices largely depends on the accuracy by which each of the inherent nanometric components can be functionally designed with predictive methods. Here, we present a rationally designed, self-assembled nanochip capable of capturing a target protein by means of pre-selected binding sites. The sensing elements comprise computationally evolved peptides, designed to target an arbitrarily selected binding site on the surface of beta-2-Microglobulin (ß2m), a globular protein that lacks well-defined pockets. The nanopatterned surface was generated by an atomic force microscopy (AFM)-based, tip force-driven nanolithography technique termed nanografting to construct laterally confined self-assembled nanopatches of single stranded (ss)DNA. These were subsequently associated with an ssDNA-peptide conjugate by means of DNA-directed immobilization, therefore allowing control of the peptide's spatial orientation. We characterized the sensitivity of such peptide-containing systems against ß2m in solution by means of AFM-based differential topographic imaging and surface plasmon resonance (SPR) spectroscopy. Our results show that the confined peptides are capable of specifically capturing ß2m from the surface-liquid interface with micromolar affinity, hence providing a viable proof-of-concept for our approach to peptide design.