The impact of COVID-19 on routine patient care from a laboratory perspective.

BACKGROUND: Globally, few studies have examined the effect of the COVID-19 pandemic on routine patient care and follow-up. OBJECTIVES: To evaluate the effect of the COVID-19 response on biochemical test requests received from outpatient departments (OPDs) and peripheral clinics serviced by the National Health Laboratory Service Chemical Pathology Laboratory at Tygerberg Hospital, Cape Town, South Africa (SA). Request volumes were used as a measure of the routine care of patients, as clinical information was not readily available. METHODS: A retrospective audit was conducted. The numbers of requests received from OPDs and peripheral clinics for creatinine, glycated haemoglobin (HbA1c), lipid profiles, thyroid-stimulating hormone (TSH), free thyroxine, free tri-iodothyronine (fT3), serum and urine protein electrophoresis, serum free light chains and neonatal total serum bilirubin were obtained from 1 March to 30 June for 2017, 2018, 2019 and 2020. RESULTS: The biggest impact was seen on lipids, creatinine, HbA1c, TSH and fT3. The percentage reduction between 1 March and 30 June 2019 and between 1 March and 30 June 2020 was 59% for lipids, 64% for creatinine and HbA1c, 80% for TSH and 81% for fT3. There was a noteworthy decrease in overall analyte testing from March to April 2020, coinciding with initiation of level 5 lockdown. Although an increase in testing was observed during June 2020, the number of requests was still lower than in June 2019. CONCLUSIONS: This study, focusing on the short-term consequences of the SA response to the COVID-19 pandemic, found that routine follow-up of patients with communicable and non-communicable diseases was affected. Future studies are necessary to evaluate the long-term consequences of the pandemic for these patient groups.

Dielectrophoresis Manipulation: Versatile Lateral and Vertical Mechanisms.

Discussing the topic of the capability of dielectrophoresis (DEP) devices in terms of the selective detection and rapid manipulation of particles based on the DEP force (FDEP) via contactless methods is challenging in medical research, drug discovery and delivery. Nonetheless, the process of the selective detection and rapid manipulation of particles via contactless DEP based on dielectric particles and the surrounding medium can reduce the effects of major issues, including physical contact with the particles and medium contamination to overcome operational difficulties. In this review, DEP microelectromechanical system (MEMS) microelectrodes with a tapered profile for the selective detection and rapid manipulation of particles were studied and compared with those of conventional designs with a straight-cut profile. The main objective of this manuscript is to review the versatile mechanism of tapered DEP MEMS microelectrodes for the purpose of selective detection and rapid manipulation. Thus, this review provides a versatile filtration mechanism with the potential for a glomerular-based membrane in an artificial kidneys' development solution for implementing engineered particles and cells by lateral attraction as well as vertical repulsion in the development of lab-on-a-chip applications. For tapered DEP MEMS microelectrodes, the scope of this study methodology involved the characterisation of DEP, modelling of the polarisation factor and the dynamic dielectric changes between the particles and medium. Comprehensive discussions are presented on the capability of tapered DEP microelectrodes to drive the selected particles and the simulation, fabrication and testing of the tapered profile. This study revealed an outstanding performance with the capability of producing two regions of high electric field intensity at the bottom and top edges of the side wall of tapered microelectrodes. Observations on particle separation mainly by the lateral attraction force of particles with positive DEP on the y-axis and vertical repulsion force of particles with negative DEP on the z-axis proved an efficient and uniform FDEP produced by tapered electrodes. In conclusion, this study confirmed the reliability and efficiency of the tapered DEP microelectrodes in the process of selective detection and rapid manipulation at a higher efficiency rate than straight-cut microelectrodes, which is significant in DEP technology applications.

Protein analysis: key to the future.

Protein analysis is crucial to elucidating the function of proteins and understanding the impact of their presence, absence and alteration. This is key to advancing knowledge about diseases, providing the opportunity for biomarker discovery and development of therapeutics. In this issue of Tech News, Nawsheen Boodhun explores the various means of protein analysis.

Translating Current Bioanalytical Techniques for Studying Corona Activity.

The recent discovery of the biological corona is revolutionising our understanding of the in vivo behaviour of nanomaterials. Accurate analysis of corona bioactivity is essential for predicting the fate of nanomaterials and thereby improving nanomedicine design. Nevertheless, current biotechniques for protein analysis are not readily adaptable for analysing corona proteins, given that their conformation, activity, and interaction may largely differ from those of the native proteins. Here, we introduce and propose tailor-made modifications to five types of mainstream bioanalytical methodologies. We specifically illustrate how these modifications can translate existing techniques for protein analysis into competent tools for dissecting the composition, bioactivity, and interaction (with both nanomaterials and the tissue) of corona formed on specific nanomaterial surfaces.

Multiplex PCR designed to differentiate species within the Candida glabrata complex / PCR multiplex diseñada para diferenciar las especies del complejo Candida glabrata

Background. No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. Aims. To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. Methods. The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. Results. The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). Conclusions. A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains (AU)

Antecedentes. No hay métodos fenotípicos disponibles para diferenciar las especies del complejo Candida glabrata. Objetivos. Diseñar un método de PCR multiplex para diferenciar las tres especies del complejo C. glabrata y usarlo para estudiar una colección de cepas identificadas anteriormente como C. glabrata. Métodos. El método fue desarrollado con base en las diferencias de la secuencia internal transcribed spacer (ITS) entre las especies. El método se validó mediante el uso de una colección de cepas incógnitas y se utilizó posteriormente para estudiar una colección de 192 cepas. Los resultados se compararon con las secuencias ITS. Resultados. El método propuesto mostró 100% de concordancia con la secuenciación de las regiones ITS y demostró ser eficaz clínica y epidemiológicamente. Se identificaron dos aislamientos de Candida bracarensis y tres de Candida nivariensis dentro de las 192 cepas identificadas fenotípicamente como C. glabrata (prevalencia de 0,93% y 1,40%, respectivamente). Conclusiones. Presentamos un método de PCR múltiplex rápido, económico y fiable. La utilidad de la metodología queda demostrada con el estudio de una gran colección de cepas de C. glabrata sensu lato (AU)

A review on preparative and semi-preparative offgel electrophoresis for multidimensional protein/peptide assessment.

Mass spectrometry (MS) techniques are commonly used for protein identification and further analysis of selected protein spots after high resolution 2-D electrophoresis. Complementary gel-free approaches have been developed during the last few years and have shown to be useful tools in modern proteomics. The development and application of various gel-free electrophoresis devices for performing protein fractionation according to the pI differences is therefore a topic of interest. This review describes the current state of isoelectric focusing (IEF) gel-free electrophoresis based on the Agilent offgel 3100 fractionator. The review includes, therefore, (i) an overview on IEF as well as other previous IEF gel-free electrophoresis developments; (ii) offgel fundamentals and future trends; (iii) advantages and disadvantages of current offgel procedures; (iv) requirements of isolated protein pellets for further offgel fractionation; (v) offgel fraction requirements to perform the second dimensional analysis by advance electrophoresis and chromatographic techniques; and (vi) effect of the offgel operating conditions on the stability of metal-protein complexes.

Guía para el estudio genético de la aniridia / Guidelines for genetic study of aniridia

Introducción: La aniridia es una enfermedad panocular con una incidencia de entre 1/50.000 a 1/100.000 nacidos vivos, que puede presentarse de forma aislada o en el contexto de un síndrome. Presenta una herencia autosómica dominante y en la mayoría de los casos está causada por mutaciones en el gen PAX6, para cuyo estudio de mutaciones se emplea una gran variedad de técnicas y metodologías de genética molecular y citogenéticas. Objetivo: Recoger los distintos aspectos de esta enfermedad y ofrecer una guía para el adecuado diagnóstico genético que ayude a un mejor manejo clínico de la misma. Desarrollo: La aniridia es una enfermedad autosómica dominante que afecta fundamentalmente al iris, pero también puede afectar a la mayoría de las estructuras oculares. Está causada principalmente por mutaciones en el gen PAX6, ubicado en la región cromosómica 11p13, que codifica para una proteína reguladora de la transcripción imprescindible en el desarrollo del ojo. El análisis genético de la aniridia es complejo y requiere tanto de técnicas de genética molecular (secuenciación, CGH-array o MLPA) como citogenéticas (cariotipo y FISH). Este estudio está indicado en todos los casos de aniridia y es importante tener en cuenta ciertas consideraciones tanto clínicas como técnicas antes de abordar su análisis y el asesoramiento genético de los pacientes y familias afectados por esta enfermedad. Conclusiones: La aplicación de técnicas de genética molecular al diagnóstico genético de la aniridia permite un mejor manejo clínico tanto de los afectados como de sus familiares(AU)

Introduction: Aniridia is a panocular disorder which occurs in 1/50,000 to 1/100,000 live births and can appear either in isolated form or in the context of a syndrome. Isolated aniridia is inherited as an autosomal dominant condition and is caused by mutations of the PAX6 gene. A variety of techniques and methodologies within molecular genetics and cytogenetics are used to study these mutations. Objective: To identify the different aspects of this disease and to provide a guide for proper genetic diagnosis leading to improved clinical management of the disease. Development: Aniridia is an autosomal dominant disease that primarily affects the iris, though it can impact most of the ocular structures. The disease is mainly caused by mutations in the PAX6 gene located on chromosome 11p13 which encodes a transcription factor that is involved in the development of the eye. Genetic analysis of aniridia is complex and requires the use of both molecular genetics and cytogenetics techniques. These procedures are indicated in all cases of aniridia. It is important bear certain clinical and technical aspects in mind prior to starting analysis or providing genetic counseling for patients and their families. Conclusions: The use of molecular genetic techniques in the genetic diagnosis of aniridia enables patients and their families to receive better clinical management(AU)

Dielectrophoresis: applications and future outlook in point of care.

Dielectrophoresis (DEP) is a label free, noninvasive, stand alone, rapid, and sensitive particle manipulation and characterization technique. Improvements in micro-electro-mechanical systems technology have enabled the biomedical applications of DEP over the past decades. By this way, integration of DEP into lab-on-a-chip systems has become achievable, creating a potential tool for point-of-care (POC) systems. DEP can be utilized in many different POC applications including early detection and prognosis of various cancer types, diagnosis of infectious diseases, blood cell analysis, and stem cell therapy. However, there are still some challenges to be resolved to have DEP-based devices available in POC market. Today, researchers have focused on these challenges to have this powerful theory as a solution for many POC applications. Here, DEP theory, cell modeling, and most common device structures are introduced briefly. Next, POC applications of DEP theory, such as cell (blood, cancer, stem, and fetal) and microorganism separation, manipulation, and enrichment for diagnosis and prognosis, are explained. Integration of DEP with other detection techniques to have more sensitive systems is summarized. Finally, future outlook for DEP-based systems are discussed with some challenges, which are currently preventing these systems to be a common tool for POC applications, and possible solutions.

[Progress in micro free flow electrophoresis].

Micro free flow electrophoresis (micro-FFE) is a continuous micro-separation or preparation technique, which has been applied in the analysis of biomolecules, such as cells, sub cell organics and proteins. In this review, the recent progresses in micro FFE are summarized, with emphasis on the design of microchips, the separation modes and the applications of micro-FFE. Furthermore, the further developments of micro-FFE are prospected.

Recent advances in electrophoretic techniques for the characterization of protein biomolecules: a poker of aces.

The four classical modes of electrophoresis of protein molecules (sodium dodecyl sulphate electrophoresis, SDS-PAGE, isoelectric focusing, IEF, and immobilized pH gradients, IPGs, two-dimensional maps, 2D, and capillary electrophoresis, CE) are here reviewed, with special emphasis on recent innovations. Thus, in the case of SDS-PAGE, a novel method, consisting in focusing SDS-protein micelles against a gradient of cationic charges grafted onto a polyacrylamide gel is presented. In the case of IEF, the recent decoding of the structure, polydispersity, molecular mass distribution and buffering properties of the soluble carrier ampholyte buffers are here discussed. In regard to two dimensional mapping, recent instrumentation for performing 2D maps in horizontal, large gel slabs (up to 30 cm × 40 cm) and in a radial format for the SDS dimension is here evaluated. Finally, in the case of CE, three major applications are presented: a thorough study of capillary IEF and of all experimental variables, a method of importance in screening of rDNA products; the possibility of running proteins and peptide separations in very acidic, amphoteric, isoelectric buffers in absence of any capillary coating; finally, the possibility of producing a facile, user friendly, covalent coating of the wall silanols via bonding of quaternarized piperazines endowed with an iodinated tail. In acidic, volatile buffers, such protein/peptide runs can be directly interfaced with mass spectrometry instrumentation.

La biodiversidad microbiana como fuente de productos de interés biotecnológico / No disponible

Introducción: La diversidad microbiana es una fuente importante de productos de interés biotecnológico, tales como antibióticos, enzimas o Polímeros 11,12. Actualmente la biodiversidad está viéndose seriamente afectada por la contaminación y por la intervención del hombre en la naturaleza. La pérdida de microorganismos no solo alterará los ecosistemas sino que supondrá la desaparición de productos de interés biotecnológico. La biodiversidad microbiana permanece aún inexplorada porque con los métodos clásicos de cultivo microbiano sólo aislamos en el laboratorio entre el 0,1 y el 10% de los microorganismos presentes en determinado ecosistema. En cambio la aplicación de herramientas moleculares permite estudiar la diversidad, estructura y la dinámica de comunidades microbianas de diferentes y complejos ambientes, así como detectar la presencia de microorganismos con interés para la industria farmacéutica y la agricultura, entre otras áreas. Objetivo: Analizar y estudiar la diversidad microbiana mediante técnicas moleculares, tanto en ambientes libres de contaminación como es el Parque natural Rambla Salada (Murcia), como en otros hábitatsc ontaminados por las actividades humanas (suelos agrícolas de Motril, Granada)(AU)

Metodología: El estudio de la diversidad microbiana requirió la puesta a punto de una estrategia molecular basada en la amplificación por PCR del gen ribosomal 16S rRNA, a partir del DNA total extraído directamente de las muestras. Posteriormente, se desarrolló la electroforesis en gel con gradiente desnaturalizante (DGGE)1 para la separación de los fragmentos del gen ribosomal 16S ya amplificados, y finalmente se procedió a la secuenciación y comparación de las secuencias obtenidas a partir de los patrones del DGGE con las existentes en las bases de datos. Por otro lado se desarrolló la técnica de hibridación in situ 7 empleando sondas fluorescentes; estas últimas fueron diseñadas para detectar la presencia de microorganismos pertenecientes a los dominios de procariotas Bacteria y Arquea. Discusión y conclusión: Mediante las herramientas moleculares hemos detectado la presencia de arqueas y bacterias no halófilas, halófilas y halotolerantes tanto en todas las zonas analizadas de Rambla Salada como algunas muestras de los suelos agrícolas de Motril. La diversidad microbiana de los suelos agrícolas de Motril es inferior a la de Rambla Salada. Se ha puesto de manifiesto la existencia de una gran diversidad microbiana en Rambla Salada compuesta fundamentalmente por procariotas pertenecientes a los phila Bacteroidetes, Cianobacteria, Proteobacteria, Firmicutes y Actinobacteria cuyos miembros no se aíslan en el laboratorio mediante las técnicas de cultivo empleadas hasta el momento. La presencia de una mayor diversidad microbiana en Rambla Salada indica que los hábitats hipersalinos son una fuente potencial de productos de interés farmacéutico(AU)

Microbial diversity is an important source of products that have potential biotechnological applications, such as antibiotics, enzymes or polymers [1, 2]. The biodiversity is seriously affected by pollution and human impact on natural environments. In this sense, reduction of biodiversity not only alters the ecosystems but also will entail the disappearance of products having biotechnological interest. Microbial biodiversity is not enough known because we have only be able so far to culture between 0.1 and 10% of the microorganisms that exist in nature. Never the less, molecular ecologytechniques represent an opportunity to study the diversity, structure and dynamics of these uncultured microorganisms, both in diverse and complex environments. They also are useful tools to detect Microorganisms that are of interest to pharmaceutical industry and agriculture, among to other areas(AU)

Therefore, the main objective of this work has been to analyse the microbial diversity in different environments using molecular methods. The habitats studied were an hypersaline soil located at Rambla Salada (Murcia), and some agricultural soils from Motril (Granada). We used PCR/DGGE to investigate the microbial diversity; this method is based on the amplification of partial 16S RNAr geneusing the total DNA extracted directly from the sample. Subsequently denaturant gradient gel electrophoresis (DGGE) [3] is developed to separate the amplified fragments of the 16S RNAr gene. Finally the sequences obtained from the DGGE patterns are compared to those available at the database. On the other hand, we used fluorescence in situ hybridization or FISH [4] to detect and quantify microorganisms be longing to the Domain Bacteria and Archaea. According to our results, non halophilic, halophilic, and halotolerant Archaea and Bacteria were present in all the areas analyzed at Rambla Salada, as well as in some samples of the agricultural soils in Motril. Microbial diversity found in the agricultural soils in Motril was lower than in Rambla Salada. Microbial community at Rambla Salada was composed of Prokaryotes belonging to the phyla Bacteroidetes, Cyanobacteria, Proteobacteria, Firmicutes and Actinobacteria, which cannot not so far isolated using methods based on traditional culture techniques(AU)

Electrophoresis today and tomorrow: Helping biologists' dreams come true.

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two-dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High-throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser-induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH-driven mobilization, provides efficient separations and on-line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single-cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single-molecule detection.

Tether forces in DNA electrophoresis.

This tutorial review introduces the concepts necessary for understanding the generation of forces on charged objects in solution by externally applied electric fields. We focus on simple, idealized cases and conceptual understanding rather than quantitative modeling. We also discuss experiments in which electrophoretic forces on DNA were directly measured. This review is aimed at readers interested in the fundamentals of electrophoresis in general, as well as those with more specific interests in DNA electrophoresis, nanopores and optical tweezers.

Dynamic computer simulations of electrophoresis: three decades of active research.

Dynamic models for electrophoresis are based upon model equations derived from the transport concepts in solution together with user-inputted conditions. They are able to predict theoretically the movement of ions and are as such the most versatile tool to explore the fundamentals of electrokinetic separations. Since its inception three decades ago, the state of dynamic computer simulation software and its use has progressed significantly and Electrophoresis played a pivotal role in that endeavor as a large proportion of the fundamental and application papers were published in this periodical. Software is available that simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. This has been employed to show the detailed mechanisms of many of the fundamental phenomena that occur in electrophoretic separations. Dynamic electrophoretic simulations are relevant for separations on any scale and instrumental format, including free-fluid preparative, gel, capillary and chip electrophoresis. This review includes a historical overview, a survey of current simulators, simulation examples and a discussion of the applications and achievements of dynamic simulation.