RESUMEN
Plasmodium vivax is the most widely distributed human malaria parasite representing 36.3% of disease burden in the South-East Asia region and the most predominant species in the region of the Americas. Recent estimates indicate that 3.3 billion of people are under risk of infection with circa 7 million clinical cases reported each year. This burden is certainly underestimated as the vast majority of chronic infections are asymptomatic. For centuries, it has been widely accepted that the only source of cryptic parasites is the liver dormant stages known as hypnozoites. However, recent evidence indicates that niches outside the liver, in particular in the spleen and the bone marrow, can represent a major source of cryptic chronic erythrocytic infections. The origin of such chronic infections is highly controversial as many key knowledge gaps remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Due to ethical and technical considerations, working with the liver, bone marrow and spleen from natural infections is very difficult. Recent advances in the development of humanized mouse models and organs-on-a-chip models, offer novel technological frontiers to study human diseases, vaccine validation and drug discovery. Here, we review current data of these frontier technologies in malaria, highlighting major challenges ahead to study P. vivax cryptic niches, which perpetuate transmission and burden.
Asunto(s)
Antimaláricos , Malaria Vivax , Malaria , Animales , Médula Ósea/parasitología , Modelos Animales de Enfermedad , Humanos , Malaria/tratamiento farmacológico , Malaria Vivax/prevención & control , Ratones , Plasmodium vivaxRESUMEN
Biomolecular gradients are widely present in multiple biological processes. Historically they were reproduced in vitro by using micropipettes, Boyden and Zigmond chambers, or hydrogels. Despite the great utility of these setups in the study of gradient-related problems such as chemotaxis, they face limitations when trying to translate more complex in vivo-like scenarios to in vitro systems. In the last 20 years, the advances in manufacturing of micromechanical systems (MEMS) had opened the possibility of applying this technology to biology (BioMEMS). In particular, microfluidics has proven extremely efficient in setting-up biomolecular gradients which are stable, controllable, reproducible and at length scales that are relevant to cells. In this chapter, we give an overview of different methods to generate molecular gradients using microfluidics, then we discuss the different steps of the pipeline to fabricate a gradient generator microfluidic device, and at the end, we show an application example of the fabrication of a microfluidic device that can be used to generate a surface-bound biomolecular gradient.
Asunto(s)
Quimiotaxis , Microfluídica , HidrogelesRESUMEN
In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term "CardioSlice." PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram-like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients.
Asunto(s)
Miocardio , Ingeniería de Tejidos , Andamios del Tejido , Técnicas de Cultivo Celular por Lotes , Fenómenos Biomecánicos , Reactores Biológicos , Diferenciación Celular , Células Cultivadas , Fenómenos Electrofisiológicos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocardio/citología , Miocardio/metabolismoRESUMEN
Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.
Asunto(s)
Filtración/métodos , Inmunoensayo/métodos , Legionella pneumophila/aislamiento & purificación , Microbiología del Agua , Legionella pneumophila/inmunología , Límite de Detección , Membranas ArtificialesRESUMEN
The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s-1, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase (HRP) bonded to the antibody, and the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The results showed that the homemade concentration system together with the developed membrane treatment protocol is able to detect E. coli cells with a limit of detection (LoD) of about 100 CFU in 100 mL. Graphical abstract Scheme of the integrated method of concentration and immunodetection of bacteria.
Asunto(s)
Escherichia coli/aislamiento & purificación , Filtración/instrumentación , Técnicas para Inmunoenzimas/instrumentación , Anticuerpos/química , Bencidinas/química , Diseño de Equipo , Infecciones por Escherichia coli/microbiología , Peroxidasa de Rábano Silvestre/química , Humanos , Límite de Detección , Microbiología del AguaRESUMEN
Legionella pneumophila is responsible for Legionnaires' disease (LD). Its detection in both environmental and clinical samples is mainly performed by culture plate method which requires up to 10days to obtain results. Nowadays, there are commercial antibodies against this bacterium, but they have not been tested against all subgroups of L. pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella bacteria. Indeed, many of these antibodies became available when only 8 serogroups of L. pneumophila had been described. We tested 7 antibodies and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L. pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the LP3IIG2 antibody detected almost all serogroups tested with lower rates of cross-reactivity, resulting in a specific sensitive antibody for the detection of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity against respiratory non-Legionella isolates, thereby contraindicating its clinical applicability.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Técnicas de Tipificación Bacteriana/métodos , Legionella pneumophila/inmunología , Enfermedad de los Legionarios/microbiología , Serotipificación/métodos , Antígenos Bacterianos/análisis , Antígenos Bacterianos/inmunología , Microbiología Ambiental , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Legionella pneumophila/aislamiento & purificación , Enfermedad de los Legionarios/inmunologíaRESUMEN
The electrokinetic transport behavior of λ-DNA (48 kbp) in 20 nm-high fused-silica nanoslits in the presence of short-chain PVP is investigated. Mobility and video data show a number of phenomena that are typical of DNA transport through gels or polymer solutions, thus indicative of rigid migration obstacles in the DNA pathway. Calculations show that a several nanometer thin layer of wall-adsorbed PVP ('nano-gel') can provide such a rigid obstacle matrix to the DNA. Such ultrathin wall-adsorbed polymer layers represent a new type of matrix for electrokinetic DNA separation.
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ADN Viral/química , Electroforesis/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Adsorción , Bacteriófago lambda/química , Bacteriófago lambda/genética , ADN Viral/análisis , Electroforesis/métodos , Técnicas Analíticas Microfluídicas/métodos , Polímeros , Dióxido de Silicio/química , Estadísticas no ParamétricasRESUMEN
Electrokinetic techniques are contact-free methods currently used in many applications, where precise handling of biological entities, such as cells, bacteria or nucleic acids, is needed. These techniques are based on the effect of electric fields on molecules suspended in a fluid, and the corresponding induced motion, which can be tuned according to some known physical laws and observed behaviours. Increasing interest on the application of such strategies in order to improve the detection of DNA strands has appeared during the recent decades. Classical electrode-based DNA electrochemical biosensors with combined electrokinetic techniques present the advantage of being able to improve the working electrode's bioactive part during their fabrication and also the hybridization yield during the sensor detection phase. This can be achieved by selectively manipulating, driving and directing the molecules towards the electrodes increasing the speed and yield of the floating DNA strands attached to them. On the other hand, this technique can be also used in order to make biosensors reusable, or reconfigurable, by simply inverting its working principle and pulling DNA strands away from the electrodes. Finally, the combination of these techniques with nanostructures, such as nanopores or nanochannels, has recently boosted the appearance of new types of electrochemical sensors that exploit the time-varying position of DNA strands in order to continuously scan these molecules and to detect their properties. This review gives an insight into the main forces involved in DNA electrokinetics and discusses the state of the art and uses of these techniques in recent years.
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Técnicas Biosensibles/métodos , ADN/análisis , Técnicas Electroquímicas/métodos , Dispositivos Laboratorio en un Chip , Algoritmos , ADN/química , ADN/aislamiento & purificación , Nanoporos , ViscosidadRESUMEN
This article presents the fabrication and characterisation of a high-speed detection micro-Coulter counter with two-dimensional (2D) adjustable aperture and differential impedance detection. The developed device has been fabricated from biocompatible and transparent materials (polymer and glass) and uses the principle of hydrodynamic focusing in two dimensions. The use of a conductive solution for the sample flux and non-conductive solutions for the focalising fluxes provides an adjustable sample flow where particles are aligned and the resistive response concentrated, consequently enhancing the sensitivity and versatility of the device. High-speed counting of 20 microm polystyrene particles and 5 microm yeast cells with a rate of up to 1,000 particles/s has been demonstrated. Two-dimensional focusing conditions have been used in devices with physical cross-sectional areas of 180 microm x 65 microm and 100 microm x 43 microm, respectively, in which particles resulted undetectable in the absence of focusing. The 2D-focusing conditions have provided, in addition, increased detection sensitivity by a factor of 1.6 as compared to 1D-focusing conditions.