The rise of big data: deep sequencing-driven computational methods are transforming the landscape of synthetic antibody design.

Synthetic antibodies (Abs) represent a category of artificial proteins capable of closely emulating the functions of natural Abs. Their in vitro production eliminates the need for an immunological response, streamlining the process of Ab discovery, engineering, and development. These artificially engineered Abs offer novel approaches to antigen recognition, paratope site manipulation, and biochemical/biophysical enhancements. As a result, synthetic Abs are fundamentally reshaping conventional methods of Ab production. This mirrors the revolution observed in molecular biology and genomics as a result of deep sequencing, which allows for the swift and cost-effective sequencing of DNA and RNA molecules at scale. Within this framework, deep sequencing has enabled the exploration of whole genomes and transcriptomes, including particular gene segments of interest. Notably, the fusion of synthetic Ab discovery with advanced deep sequencing technologies is redefining the current approaches to Ab design and development. Such combination offers opportunity to exhaustively explore Ab repertoires, fast-tracking the Ab discovery process, and enhancing synthetic Ab engineering. Moreover, advanced computational algorithms have the capacity to effectively mine big data, helping to identify Ab sequence patterns/features hidden within deep sequencing Ab datasets. In this context, these methods can be utilized to predict novel sequence features thereby enabling the successful generation of de novo Ab molecules. Hence, the merging of synthetic Ab design, deep sequencing technologies, and advanced computational models heralds a new chapter in Ab discovery, broadening our comprehension of immunology and streamlining the advancement of biological therapeutics.

Revolutionizing Synthetic Antibody Design: Harnessing Artificial Intelligence and Deep Sequencing Big Data for Unprecedented Advances.

Synthetic antibodies (Abs) represent a category of engineered proteins meticulously crafted to replicate the functions of their natural counterparts. Such Abs are generated in vitro, enabling advanced molecular alterations associated with antigen recognition, paratope site engineering, and biochemical refinements. In a parallel realm, deep sequencing has brought about a paradigm shift in molecular biology. It facilitates the prompt and cost-effective high-throughput sequencing of DNA and RNA molecules, enabling the comprehensive big data analysis of Ab transcriptomes, including specific regions of interest. Significantly, the integration of artificial intelligence (AI), based on machine- and deep- learning approaches, has fundamentally transformed our capacity to discern patterns hidden within deep sequencing big data, including distinctive Ab features and protein folding free energy landscapes. Ultimately, current AI advances can generate approximations of the most stable Ab structural configurations, enabling the prediction of de novo synthetic Abs. As a result, this manuscript comprehensively examines the latest and relevant literature concerning the intersection of deep sequencing big data and AI methodologies for the design and development of synthetic Abs. Together, these advancements have accelerated the exploration of antibody repertoires, contributing to the refinement of synthetic Ab engineering and optimizations, and facilitating advancements in the lead identification process.

Current advancements in B-cell receptor sequencing fast-track the development of synthetic antibodies.

Synthetic antibodies (Abs) are a class of engineered proteins designed to mimic the functions of natural Abs. These are produced entirely in vitro, eliminating the need for an immune response. As such, synthetic Abs have transformed the traditional methods of raising Abs. Likewise, deep sequencing technologies have revolutionized genomics and molecular biology. These enable the rapid and cost-effective sequencing of DNA and RNA molecules. They have allowed for accurate and inexpensive analysis of entire genomes and transcriptomes. Notably, via deep sequencing it is now possible to sequence a person's entire B-cell receptor immune repertoire, termed BCR sequencing. This procedure allows for big data explorations of natural Abs associated with an immune response. Importantly, the identified sequences have the ability to improve the design and engineering of synthetic Abs by offering an initial sequence framework for downstream optimizations. Additionally, machine learning algorithms can be introduced to leverage the vast amount of BCR sequencing datasets to rapidly identify patterns hidden in big data to effectively make in silico predictions of antigen selective synthetic Abs. Thus, the convergence of BCR sequencing, machine learning, and synthetic Ab development has effectively promoted a new era in Ab therapeutics. The combination of these technologies is driving rapid advances in precision medicine, diagnostics, and personalized treatments.

Encuesta colombiana de neurología 2016-2020 / Colombian neurology survey 2016-2020

RESUMEN INTRODUCCIÓN: La práctica de la neurología como especialidad clínica es relativamente reciente en Colombia, a pesar de que esta área ha mostrado progresos académicos significativos; la información sociodemográfica es limitada. OBJETIVO: Describir las características sociodemográficas de los neurólogos laboralmente activos en Colombia. METODOLOGÍA: Estudio descriptivo en dos periodos. La información se obtuvo mediante encuestas autodiligenciadas a los asistentes al Congreso Nacional de Neurología del año 2016; la del 2020 se recolectó empleando cuestionarios en línea a través de formularios Google. RESULTADOS: Se contabilizaron 549 neurólogos laboralmente activos en el territorio colombiano. El análisis de las muestras 2016 y 2020 mostró que la mayor proporción de estos especialistas se concentraba en Bogotá (45,4 %), Medellín (13,4 %) y Cali (8,4 %), con una ocupación escasa en ciudades no capitales. La comparación de horas laborales e ingresos económicos al analizar 2016 y 2020 no mostró diferencias. El mayor tiempo de ejercicio se correlacionó con mayores ingresos, tanto en el 2016 (p < 0,001) como en el 2020 (p < 0,01). CONCLUSIONES: Excepto por el incremento en la población de nuevos neurólogos, las características socio-demográficas de los neurólogos en Colombia se mantienen sin variaciones al comparar los años 2016 y 2020.

ABSTRACT INTRODUCTION: Neurology practice is relatively recent in Colombia. Even though this area has shown significant academic advances, information regarding sociodemographic conditions is limited. OBJECTIVE: To describe sociodemographic characteristics of neurologists who are currently active in Colombia. METHODS: Descriptive study over two time periods. The information was obtained by means of self-administered surveys to the neurologists attending the neurology national congress in 2016. In 2020, data was collected by means of on-line questionnaires using google forms. RESULTS: The sample included 549 neurologists. The largest proportion of these specialists were located in Bogotá (45.4 %), Medellín (13.4 %) and Cali (8.4 %). After comparing working hours per week and income we did not identify differences between these 2 years. The time of work experience was correlated with economic income both in 2016 (p<0.001) as in 2020 (p<0.01). CONCLUSION: Except for increasing number of neurologists of recent graduation, the sociodemographic characteristics of Colombian neurologists remain stable when comparing 2016 and 2020.

Tétanos generalizado: reporte de caso y análisis clínico para su diferenciación con otras entidades como las crisis epilépticas / Tetanus as a seizure crisis simulator in a 70-year-old patient, case report

RESUMEN INTRODUCCIÓN: El tétanos es una enfermedad que afecta el sistema nervioso. Su presentación clínica se caracteriza por espasmos musculares en respuesta a la liberación de la neurotoxina producida por la formación de esporas de la bacteria Clostridium tetani. DESCRIPCIÓN DEL CASO: Presentamos el caso de un hombre de 70 años que luego de una caída presentó una herida en la región ocular. Al ingreso se evidenciaron signos de infección local y contracción involuntaria en los músculos maseteros, con imposibilidad de apertura oral. Posteriormente, presentó insuficiencia respiratoria, contracciones generalizadas y necesidad de traslado a unidad de cuidado intensivo. Debido a que entre los diagnósticos diferenciales se encontraba la presencia de crisis epilépticas motoras, se hicieron estudios complementarios para descartar esta posibilidad. DISCUSIÓN: El diagnóstico del tétanos es clínico, es importante sospecharlo en pacientes con antecedentes de lesión en piel e inmunización inadecuada. Por su amplia presentación clínica, puede llevar a confusión con otras patologías. Entre los diagnósticos diferenciales están las crisis epilépticas, sin embargo, el tétano no cumple con las características semiológicas, no compromete el estado de conciencia y no progresa a estado epiléptico, asociado con la normalidad de estudios complementarios como las neuroimágenes, el estudio de líquido cefalorraquídeo y el registro electroencefalográfico. CONCLUSIÓN: El tétanos es una enfermedad altamente prevenible y un reto diagnóstico para el profesional de la salud por su amplio debut de síntomas. Por ello, en el abordaje diagnóstico es importante reconocer los diagnósticos diferenciales, teniendo como base la historia clínica, lo que permite un diagnóstico temprano y oportuno.

ABSTRACT INTRODUCTION: Tetanus is a disease that affects the nervous system and its clinical presentation is characterized by muscle spasms caused by the release of a neurotoxin produced by the formation of spores of the Clostridium tetani bacteria. CASE DESCRIPTION: We present the case of a 70-year-old man who after a fall, presented an injury to the ocular region. On admission, signs of local infection and involuntary contraction of the masseter muscles were evident, with impossibility of oral opening. Subsequently, he presented respiratory failure, generalized contractions and transfer to the intensive care unit, due to its similarity to convulsive events, pathology at the level of the central nervous system is suspected, for which it requires complementary studies and clinical analysis to rule it out. DISCUSSION: The diagnosis of tetanus is clinical, it is important to suspect it in patients with a history of skin lesions and inadequate immunization, due to its extensive clinical presentation, it can lead to confusion with other pathologies. Among the differential diagnoses are epileptic seizures, however, tetanus does not meet the semiological characteristics, does not compromise the state of consciousness and does not progress to status epilepticus, associated with the normality of complementary studies such as neuroimaging, cerebrospinal fluid study and registry electroencephalographic. CONCLUSION: Tetanus is a highly preventable disease and a diagnostic challenge for the health professional due to its wide onset of symptoms. That is why the diagnostic approach is important to recognize the differential diagnoses based on the clinical history, which allows an early and timely diagnosis.

USP10 Promotes Fibronectin Recycling, Secretion, and Organization.

Purpose: Integrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFß activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs). Methods: Primary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry. Results: USP10 overexpression increased α5ß1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7-2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5ß1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN. Conclusions: Our data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.

Inhibition of Cancer Cell Adhesion, Migration and Proliferation by a Bispecific Antibody that Targets two Distinct Epitopes on αv Integrins.

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFß) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFß. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvß1, αvß3 and αvß5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.

Síndrome de burnout en los neurólogos colombianos: prevalência y factores asociados / Burnout syndrome in Colombian neurologists: prevalence and associated factors

RESUMEN INTRODUCCIÓN: El síndrome de burnout es una condición de prevalência creciente que afecta la calidad de vida y los resultados laborales de quienes lo padecen. OBJETIVO: Describir la prevalencia y factores asociados del síndrome de burnout en neurólogos colombianos. METODOLOGÍA: Mediante encuesta autoadministrada se obtuvo información de 119 neurólogos laboralmente activos en Colombia. Se incluyeron datos correspondientes a variables sociodemográficas junto con la escala Maslasch Burnout Inventory. Para calcular la correlación estadística de variables se utilizó regresión logística. RESULTADOS: El síndrome de burnout se determinó en el 49,6 % de los entrevistados (afectación de 2 o más dimensiones). Esta condición se correlacionó con el sexo femenino (P=0,036), el número de horas trabajadas por semana (P=0,040) y la frecuencia de satisfacción con el trabajo (P<0,001). La práctica de actividades de esparcimiento fue estadísticamente significativa (P=0,024) como factor protector. CONCLUSIÓN: El síndrome de burnout es una condición prevalente en los neurólogos en Colombia. Esta información es útil para la creación de políticas encaminadas a mejorar las condiciones del ejercicio de esta especialidad en nuestro país.

SUMMARY INTRODUCTION: Burnout syndrome is a condition of increasing prevalence that affects quality of life and labor outcomes. OBJECTIVE: To describe the prevalence and factors related to burnout syndrome in Colombian neurologists. METHODOLOGY: By mean of a self-administered survey we obtained information from 119 neurologists currently working in Colombia. Sociodemographic and Maslasch Burnout Inventory data were collected. To calculate statistical correlation of variables related to the syndrome a logistic regression model was used. RESULTS: Burnout syndrome was determined in 49.6% of interviewed neurologists (2 or more affected dimensions).This condition was related to female gender (P=0.036), number of hours worked weekly (P=0.040) and level of work satisfaction (P<0.001). Having a hobby was determined as protector for burnout (P=0.024). CONCLUSION: Burnout syndrome is a prevalent condition in Colombian neurologists. This information should be considered for designing policies directed to better labor conditions for this specialty in our country.

CellectSeq: In silico discovery of antibodies targeting integral membrane proteins combining in situ selections and next-generation sequencing.

Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.

The EphA2 and cancer connection: potential for immune-based interventions.

The Eph (erythropoietin-producing human hepatocellular) receptors form the largest known subfamily of receptor tyrosine kinases. These receptors interact with membrane-bound ephrin ligands via direct cell-cell interactions resulting in bi-directional activation of signal pathways. Importantly, the Eph receptors play critical roles in embryonic tissue organization and homeostasis, and in the maintenance of adult processes such as long-term potentiation, angiogenesis, and stem cell differentiation. The Eph receptors also display properties of both tumor promoters and suppressors depending on the cellular context. Characterization of EphA2 receptor in regard to EphA2 dysregulation has revealed associations with various pathological processes, especially cancer. The analysis of various tumor types generally identify EphA2 receptor as overexpressed and/or mutated, and for certain types of cancers EphA2 is linked with poor prognosis and decreased patient survival. Thus, here we highlight the role of EphA2 in malignant tissues that are specific to cancer; these include glioblastoma multiforme, prostate cancer, ovarian and uterine cancers, gastric carcinoma, melanoma, and breast cancer. Due to its large extracellular domain, therapeutic targeting of EphA2 with monoclonal antibodies (mAbs), which may function as inhibitors of ligand activation or as molecular agonists, has been an oft-attempted strategy. Therefore, we review the most current mAb-based therapies against EphA2 expressing cancers currently in pre-clinical and/or clinical stages. Finally, we discuss the latest peptides and cyclical-peptides that function as selective agonists for EphA2 receptor.

Critical role of EphA3 in cancer and current state of EphA3 drug therapeutics.

The erythropoietin-producing human hepatocellular (Eph) receptors are transmembrane glycoprotein members of the tyrosine kinase receptors family. The Ephs may bind to various ephrin ligands resulting in the phosphorylation of their tyrosine kinase domain and the activation of the Eph receptor. In this review we focus on EphA3, one receptor of the 14 different Ephs, as it carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. The loss of EphA3 regulation is correlated with various human malignancies, the most notable being cancer. This receptor is overexpressed and/or mutated in multiple tumors, and is also associated with poor prognosis and decreased survival in patients. Here we highlight the role of EphA3 in normal and malignant tissues that are specific to cancer; these include hematologic disorders, gastric cancer, glioblastoma multiforme, colorectal cancer, lung cancer, renal cell carcinoma, and prostate cancer. Moreover, various anticancer agents against EphA3 have been developed to either inhibit its kinase domain activity or to function as agonists. Thus, we examine the most potent small molecule drugs and mAb-based therapeutics against EphA3 that are currently in pre-clinical or clinical stages.

Epidermal growth factor receptor (EGFR) involvement in epithelial-derived cancers and its current antibody-based immunotherapies.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that is part of the family of tyrosine kinase receptors. The binding of EGFR to its cognate ligands leads to its autophosphorylation and subsequent activation of the signal transduction pathways involved in regulating cellular proliferation, differentiation, and survival. Accordingly, this receptor carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. Correspondingly, the loss of EGFR regulation results in many human diseases, with the most notable cancer. This receptor is overexpressed and/or mutated in multiple epithelial-derived tumors, and associated with poor prognosis and survival in cancer patients. Here, we discuss in detail the role of EGFR in specific epithelial-derived cancer pathologies; these include lung cancer, colorectal cancer, and squamous cell carcinomas. The development of multiple anticancer agents against EGFR diminished the progression and metastasis of tumors. Some of the most versatile therapeutic anti-EGFR agents include the monoclonal antibodies (mAbs), demonstrating success in clinical settings when used in combination with cytotoxic treatments, such as chemotherapy and/or radiation. We thus discuss the development and application of two of the most notable therapeutic mAbs, cetuximab, and panitumumab, currently utilized in various EGFR-related epithelial cancers.

In situ antibody phage display yields optimal inhibitors of integrin α11/ß1.

Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/ß1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/ß1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/ß1. Conversely, none of the antibodies selected for binding to purified integrin-α11/ß1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/ß1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/ß1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

High-Throughput Generation of In Silico Derived Synthetic Antibodies via One-step Enzymatic DNA Assembly of Fragments.

Phage-display technology offers robust methods for isolating antibody (Ab) molecules with specificity for different target antigens. Recent advancements couple Ab selections with in silico strategies, such as predictive computational models or next-generation sequencing metadata analysis of Ab selections. These advancements result in enhanced Ab clonal diversities with potential for enlarged epitope coverage of the target antigen. A current limitation however, is that de novo Ab sequences must undergo DNA gene synthesis, and subsequent expression as Ab proteins for downstream validations. Due to the high costs and time for commercially generating large sets of DNA genes, we report a high-throughput platform for the synthesis of in silico derived Ab clones. As a proof of concept we demonstrate the simultaneous synthesis of 96 unique Abs with varied lengths and complementary determining region compositions. Each of the 96 Ab clones undergoes a one-step enzymatic assembly of distinct DNA fragments that combine into a circularized Fab expression plasmid. This strategy allows for the rapid and efficient synthesis of 96 DNA constructs in a 3 day window, and exhibits high percentage fidelity-greater than 93%. Accordingly, the synthesis of Ab DNA constructs as Fab expression plasmids allow for rapid execution of downstream Ab protein validations, with potential for implementation into high-throughput Ab protein characterization pipelines. Altogether, the platform presented here proves rapid and also cost-effective, which is important for labs with limited resources, since it utilizes standard laboratory equipment and molecular reagents.

Fluorogen-Activating Proteins: Next-Generation Fluorescence Probes for Biological Research.

Since their discovery, fluorescent probes have found widespread use in biological research. Over time, multiple next-generation probes increased the fluorescence catalog by offering novel capabilities of detection that have been previously difficult or lacking with conventional probes. One of such probes is called a fluorogen-activating protein (FAP). These are bimodular sensors, composed of a single-chain antibody that exhibits high-affinity and selectivity for small-molecule fluorogens. Because fluorogens are inherently nonfluorescent unless sterically restricted, upon the formation of the noncovalent FAP-fluorogen complex the fluorogen module emits fluorescence when excited by light. More interestingly, these bimodular sensors permit improvement of their biophysical properties. For instance, the fluorescence spectra and environmental sensing capabilities of fluorogens may be altered by the method of chemical modification at the fluorogen structural level. Also, optimizations of the single-chain antibody scaffold, via amino acid substitutions at the selectivity regions, may improve the detection brightness and affinities of fluorogens; this may also improve the biophysical stability of FAPs in different cellular environments. Additionally, when utilized as biological discovery probes, FAP biosensors exhibit functional activity as genetic fusion tags with cellular proteins; this results in high fluorescent sensitivities of cell surface and intracellular targets. Also, FAPs allow the monitoring of cellular traffic of surface receptors by fluorescence methods of real-time color switching, or signal onset and offset. They find application as biological probes integrated into biomaterials, or as soluble affinity reagents for whole live animal studies. Overall, this noncovalent activation of fluorogen particles results in advanced strategies of fluorescence detection.

A High-Throughput Platform for the Generation of Synthetic Ab Clones by Single-Strand Site-Directed Mutagenesis.

Current developments in meta-data analysis and predictive computational models offer alternative routes for the identification of antibodies. In silico-based technologies and NGS data analysis from Ab phage-display selections offer expanded selections of Ab candidates. Accordingly, the identified de novo Abs with predicted selectivity for a target antigen must undergo rapid gene synthesis for downstream Ab characterizations. Here we describe a high-throughput strategy for the generation of synthetic Ab clones for expression as Fab proteins in Escherichia coli. Our approach utilizes simultaneous single-stranded site-directed mutagenesis of diversified Ab regions of a phagemid template with engineered complementary determining regions that contain multiple stop codon and restriction enzyme sites. Subsequently, we perform rapid screening of Ab DNA clones for correct gene assemblies by high-throughput Ab-phage protein expression screens. Identified sequences are corroborated by Sanger DNA sequencing analysis. In summary, our work describes a rapid and cost-effective platform for the high-throughput synthesis of synthetic Ab genes as Fab proteins for implementation into downstream protein validation pipelines.

The perception of parents and guardians about the health and quality of life of their adolescent children enrolled in school / Percepciones de padres o acudientes sobre la salud y calidad de vida de sus hijos adolescentes escolarizados / Percepções de pais ou responsáveis sobre a saúde e a qualidade de vida de seus filhos adolescentes escolarizados

Introduction. The perception of parents or guardians about their children's quality of life allows to assess the children's health, bearing in mind the abilities to fully participate in age-appropriate physical, social and psychosocial functions and activities. Research on quality of life (QL) and children's quality of life as regards their health is a field that, although recent, has made significant progress in the past years. Various measurement instruments have been developed from a quantitative perspective to assess it, but few studies analyze the perceptions from a qualitative focus. Objective. To recognize the perception of parents and guardians about the health and quality of life of their adolescent children. Methodology. Qualitative study with the focus group technique in which nine parents or caregivers participated. A focus group was created and the interview was recorded and transcribed. Results. Eleven categories that cover the health and quality of life in the stage of adolescents enrolled in school were detected. Dimensions appeared that are not reported by health-related quality of life instruments, such as spirituality and technology. Discussion. In the area of spirituality, studies have demonstrated a strong positive correlation between parents, religiosity and the reduction of risky conducts in their children. Conclusions. Addressing parents or caregivers helped identify other aspects of health and quality of life that can affect their adolescent children. [Jaimes-Valencia ML, Fajardo-Nates S, Argüello JF, Mejía-Arciniegas CN, Rojas-Arenas LC, Gallo-Eugenio LM, León- Santos NR. The perception of parents and guardians about the health and quality of life of their adolescent children enrolled in school. MedUNAB. 2019;21(3):314-333. doi: 10.29375/01237047.2736]

Introducción. La percepción que tienen los padres o acudientes sobre la calidad de vida de sus hijos, permite realizar una valoración de la salud de los niños teniendo en cuenta las habilidades de participar plenamente en funciones y actividades físicas, sociales y psicosociales apropiadas para la edad. La investigación en la Calidad de Vida (CV) y calidad de vida relacionada con la salud en niños es un campo que, aunque reciente, ha tenido progresos importantes en los últimos años; para su valoración se han creado varios instrumentos de medición desde la perspectiva cuantitativa, pero son escasos los estudios que analizan las percepciones desde un enfoque cualitativo. Objetivo. Reconocer las percepciones que tienen los padres o acudientes sobre la salud y calidad de vida de sus hijos adolescentes. Metodología. Estudio cualitativo con la técnica de grupo focal en la que participaron 9 padres o acudientes. Se realizó un grupo focal, se grabó y transcribió la entrevista. Resultados. Se detectaron 11 categorías que hacen parte de la salud y calidad de vida en la etapa de los adolescentes escolarizados. Surgieron dimensiones que no son reportadas por instrumentos de calidad de vida relacionada con la salud como es la espiritualidad y la tecnología. Respecto a la espiritualidad, estudios han demostrado una correlación positiva fuerte entre los padres, religiosidad y reducción de conductas de riesgo de sus hijos/as. Conclusiones. Abordar a los padres o acudientes permitió identificar otros aspectos de la salud y calidad de vida que pueden afectar a sus hijos en la adolescencia. [Jaimes-Valencia ML, Fajardo-Nates S, Argüello JF, Mejía-Arciniegas CN, Rojas-Arenas LC, Gallo- Eugenio LM, León-Santos NR. Percepciones de padres o acudientes sobre la salud y calidad de vida de sus hijos adolescentes escolarizados. MedUNAB. 2019;21(3):314- 333. doi: 10.29375/01237047.2736]

Introdução. A percepção que os pais ou responsáveis têm sobre a qualidade de vida de seus filhos permite uma avaliação da saúde das crianças, considerando as habilidades para participar plenamente de eventos e atividades físicas, sociais e psicossociais adequadas à idade. A pesquisa sobre qualidade de vida (QV) e qualidade de vida relacionada à saúde em crianças é um campo que, apesar de recente, teve importantes avanços nos últimos anos. Para sua avaliação, tem sido criados vários instrumentos de medição da perspectiva quantitativa, mas há poucos estudos que analisam percepções a partir de uma perspectiva qualitativa. Objetivo. Reconhecer as percepções que os pais ou responsáveis têm sobre a saúde e a qualidade de vida de seus filhos adolescentes. Metodologia. Estudo qualitativo com a técnica de grupo focal em que participaram nove pais ou responsáveis. Foi organizado um grupo focal, e a entrevista foi gravada e transcrita. Resultados. Foram detectadas onze categorias que fazem parte da saúde e qualidade de vida na fase de adolescentes escolarizados. Emergiram dimensões que não são relatadas através de instrumentos de qualidade de vida relacionada à saúde, como espiritualidade e tecnologia. Discussão. Em relação à espiritualidade, estudos têm mostrado uma forte correlação positiva entre pais, religiosidade e redução de comportamentos de risco de seus filhos. Conclusões. Dirigirse aos pais ou responsáveis que participaram, ajudou a identificar outros aspectos da saúde e da qualidade de vida que podem afetar seus filhos durante a adolescência. [Jaimes-Valencia ML, Fajardo- Nates S, Argüello JF, Mejía-Arciniegas CN, Rojas-Arenas LC, Gallo-Eugenio LM, León-Santos NR. Percepções de pais ou responsáveis sobre a saúde e a qualidade de vida de seus filhos adolescentes escolarizados. MedUNAB. 2019;21(3):314-333. doi: 10.29375/01237047.2736]

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection.

A recently described fluorescence biosensor platform utilizes single-chain Fv (scFvs) that selectively bind and activate fluorogen molecules. In this report we investigated the display of tandem scFv biosensors at the surface of mammalian cells with the aim of advancing current fluorescence detection strategies. We initially screened different peptide linkers to separate each scFv unit, and discovered that tandem proteins joined by either flexible or α-helical linkers properly fold and display at the surface of mammalian cells. Accordingly, we performed a combinatorial scFv-dimer study and identified that fluorescence activation correlated with the cellular location (membrane distal versus proximal) and selections of the different scFvs. Furthermore, in vitro measurements showed that the stability of each scFv monomer unit influenced the folding and cell surface activities of tandem scFvs. Additionally, we investigated the absence or poor signals from some scFv-dimer combinations and discovered that intramolecular and intermolecular scFv chain mispairings led to protein misfolding and/or secretory-pathway-mediated degradation. Furthermore, when tandem scFvs were utilized as fluorescence reporter tags with surface receptors, the biosensor unit and target protein showed independent activities. Thus, the live cell application of tandem scFvs permitted advanced detection of target proteins via fluorescence signal amplification, Förster resonance energy transfer resulting in the increase of Stokes shift and multi-color vesicular traffic of surface receptors.