Glycosylation-on-a-Chip: A Flow-Based Microfluidic System for Cell-Free Glycoprotein Biosynthesis.

In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells.

Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the "glycan code") and its application (e.g., biomanufacturing high-value glycomolecules on demand).

Manifestaciones clínicas y laboratoriales en el lupus eritematoso sistémico - LES / Clinical and laboratory manifstations in systemic Lupus erythematosus

El Lupus eritematoso sistémico (LES) es una enfermedad autoinmune compleja que se caracteriza por su capacidad de afectar a diversos órganos, lo que determina las diferentes manifestaciones clínicas objetivadas durante la evolución de la enfermedad. De forma asociada se ha descrito que estas manifestaciones presentan una variación geográfica o étnica, siendo por lo general menos grave en pacientes con ascendencia europea que en aquellos que presentan ascendencia africana, asiática o hispana. Alteraciones, tanto del sistema inmune adaptativo (células T y B) como del innato (Toll like receptorx-TLR), contribuyen al desarrollo del LES. Las células B tienen su papel en la producción de los autoanticuerpos (i.e. anticuerpos anti-ADN y anticuerpos anti-nucleosoma) y de determiandas citocinas. Las pruebas de laboratorio son de gran valor cuando se evalúa a un paciente con sospecha de enfermedad autoinmune. Los resultados pueden confirmar el diagnóstico, estimar la severidad de la enfermedad, evaluar el pronóstico y son de suma utilidad para el seguimiento de la actividad del LES.

Systemic Lupus Erythematosus (SLE) is a complex autoimmnune disease characterizedby its ability to affect different organs, which determines different clinical manifestationsobserved during the course of the disease. It has been described that thesemanifestations have geographic or ethnic varations being generally less serious in patientsof European descent than in those with African, Asian or Hispanic descents. Alterations ofboth the adaptative (T and B cells) and innate (Toll like receptorx-TLR) immnune systemscontribute to the development of SLE. B cells have a role in the production of autoantibodies(i.e. anti-DNA and anti-nucleosome antibodies) and some cytokines. Laboratorytests are invaluable when evaluating a patient with suspected autoimmune disease. Theresults can confirm the diagnosis, estimate the severity of the disease, assess prognosisand are extremely useful for monitoring the activity of SLE.

Déficit de vitamina D en pacientes post-menopáusicas y su relación con el metabolismo fosfocálcico y la osteoporosis / Vitamin D deficiency in post-menopausal patients and its relation with the mineral metabolism and the osteoporosis

La influencia de la hipovitaminosis D en la mujer post-menopáusica constituye un tema de gran importancia por las implicancias en el metabolismo fosfo-cálcico y su posible asociación en el desarrollo de otros tipos de patologías. Es por eso que el presente estudio tiene por objetivo conocer la prevalencia de la hipovitaminosis D en una población de mujeres post-menopáusicas y su asociación con los cambios en el metabolismo fosfocálcico y con el desarrollo de la osteoporosis. Se incluyó 67 mujeres post-menopáusicas procedentes de una consulta ambulatoria de reumatología. Se consideraron las siguientes variables clínicas (i.e. edad, peso), laboratorio (i.e. concentraciones de calcio, fosforo y PTH) y la presencia o ausencia de osteopenia u osteoporosis. El valor de la media de la edad de las pacientes fue de 66 ± 11,29 años y las concentraciones de vitamina D inferior a 30 ng/ml se observó en 50 (74,6%) pacientes. La osteopenia u osteoporosis se observó en una parte importante de nuestros pacientes. No se observó una correlación significativa entre las concentraciones de vitamina D y las concentraciones de calcio y fósforo. Se observó una correlación negativa en relación a las concentraciones de PTH (P= 0,049). Las pacientes con osteoporosis u osteopenia presentan con frecuencia hipovitaminosis D. Es por eso que existe la necesidad de realizar una detección y tratamiento temprano a fin de evitar las graves complicaciones que podrían acompañar a la pérdida de densidad ósea en este grupo de pacientes.