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Histones are basic proteins with an isoelectric point around 11. It has been shown that the level of plasma circulating histones increases significantly during sepsis, and circulating free histones are associated with sepsis severity and mortality. It was found that the median plasma total free histone concentration of sepsis ICU non-survivors is higher compared to survivors. Therefore, histone concentration can serve as a prognostic indicator and there is a need for a simple, low-cost, and rapid method for measuring histone levels. In this work, we have developed a microfluidic device containing an isoelectric membrane made of dehydrated agarose gel of a specific pH embedded in a porous membrane for isoelectric trapping of histones rapidly. Although isoelectric gates have been used for trapping proteins before, they have to be introduced at the time of the experiment. Here, we show that isoelectric gates formed by gels loaded in a scaffold can be integrated directly into the fabrication process flow, dehydrated for storage, and rehydrated during the experiment and still function effectively to achieve isoelectric trapping. A low-cost and rapid microfabrication technique, xurography, was used for agarose integration and device fabrication. The integrated device was tested with samples containing buffered histone, histone in the presence of high-concentration bovine serum albumin (BSA), and histone spiked in blood plasma. The results show that the device can be used to distinguish between survivors and non-survivors of sepsis in less than 10 min, making it suitable as a point-of-care device for sepsis prognosis.
Assuntos
Histonas , Sepse , Humanos , Sefarose , Prognóstico , Sepse/diagnóstico , Dispositivos Lab-On-A-ChipRESUMO
Respiratory syncytial virus (RSV) is one of the major pathogens responsible for lower respiratory tract infections (LRTI) in young children, the elderly, and the immunosuppressed. Currently, there are no antiviral drugs or vaccines available that effectively target RSV infections, proving a significant challenge in regards to prevention and treatment. An in-depth understanding of the host-virus interactions that underlie assembly and budding would inform new targets for antiviral development.Current research suggests that the polymerised form of actin, the filamentous or F-actin, plays a role in RSV assembly and budding. Treatment with cytochalasin D, which disrupts F-actin, has been shown to inhibit virus release. In addition, the actin cytoskeleton has been shown to interact with the RSV matrix (M) protein, which plays a central role in RSV assembly. For this reason, the interaction between these two components is hypothesised to facilitate the movement of viral components in the cytoplasm and to the budding site. Despite increases in our knowledge of RSV assembly and budding, M-actin interactions are not well understood. In this review, we discuss the current literature on the role of actin cytoskeleton during assembly and budding of RSV with the aim to integrate disparate studies to build a hypothetical model of the various molecular interactions between actin and RSV M protein that facilitate RSV assembly and budding.
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Sepsis, a life-threatening condition resulting from a failing host response to infection, causes millions of deaths annually, necessitating rapid and simple prognostic assessments. A variety of genomic and proteomic biomarkers have been developed for sepsis. For example, it has been shown that the level of plasma cell-free DNA (cfDNA) and circulating histones increases considerably during sepsis, and they are linked with sepsis severity and mortality. Developing a diagnostic tool that is capable of assessing such diverse biomarkers is challenging as the detection methodology is quite different for each. Here, a fully integrated microfluidic device capable of detecting a genomic biomarker (cfDNA) and a proteomic biomarker (total circulating histones) using a common detection platform has been demonstrated. The microfluidic device utilizes dehydrated agarose gates loaded with pH-specific agarose to electrophoretically trap cfDNA and histones at their respective isoelectric points. It also incorporates fluorescent dyes within the device, eliminating the need for off-chip sample preparation and allowing the direct testing of plasma samples without the need for labeling DNA and histones with fluorescent dyes beforehand. Xurography, which is a low-cost and rapid method for fabrication of microfluidics, is used in all the fabrication steps. Experimental results demonstrate the effective accumulation and separation of cfDNA and histones in the agarose gates in a total processing time of 20 min, employing 10 and 30 Volts for cfDNA and histone accumulation and detection, respectively. The device can potentially be used to distinguish between the survivors and non-survivors of sepsis. The integration of the detection of both biomarkers into a single device and dye immobilization enhances its clinical utility for rapid point-of-care assessment of sepsis prognosis.
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Human Rhinoviruses (RV) are a major cause of common colds and infections in early childhood and can lead to subsequent development of asthma via an as yet unknown mechanism. Asthma is a chronic inflammatory pulmonary disease characterized by significant airway remodeling. A key component of airway remodeling is the transdifferentiation of airway epithelial and fibroblast cells into cells with a more contractile phenotype. Interestingly, transforming growth factor-beta (TGF-ß), a well characterized inducer of transdifferentiation, is significantly higher in airways of asthmatics compared to non-asthmatics. RV infection induces TGF-ß signaling, at the same time nucleoporins (Nups), including Nup153, are cleaved by RV proteases disrupting nucleocytoplasmic transport. As Nup153 regulates nuclear export of SMAD2, a key intermediate in the TGF-ß transdifferentiation pathway, its loss of function would result in nuclear retention of SMAD2 and dysregulated TGF-ß signaling. We hypothesize that RV infection leads to increased nuclear SMAD2, resulting in sustained TGF-ß induced gene expression, priming the airway for subsequent development of asthma. Our hypothesis brings together disparate studies on RV, asthma and Nup153 with the aim to prompt new research into the role of RV infection in development of asthma.
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Hydrogels are a critical component of many microfluidic devices. They have been used in cell culture applications, biosensors, gradient generators, separation microdevices, micro-actuators, and microvalves. Various techniques have been utilized to integrate hydrogels into microfluidic devices such as flow confinement and gel photopolymerization. However, in these methods, hydrogels are typically introduced in post processing steps which add complexity, cost, and extensive fabrication steps to the integration process and can be prone to user induced variations. Here, we introduce an inexpensive method to locally integrate hydrogels into microfluidic devices during the fabrication process without the need for post-processing. In this method, porous and fibrous membranes such as electrospun membranes are used as scaffolds to hold gels and they are patterned using xurography. Hydrogels in various shapes as small as 200 µm can be patterned using this method in a scalable manner. The electrospun scaffold facilitates drying and reconstitution of these gels without loss of shape or leakage that is beneficial in a number of applications. Such reconstitution is not feasible using other hydrogel integration techniques. Therefore, this method is suitable for long time storage of hydrogels in devices which is useful in point-of-care (POC) devices. This hydrogel integration method was used to demonstrate gel electrophoretic concentration and quantification of short DNA (150 bp) with different concentrations in rehydrated agarose embedded in electrospun polycaprolactone (PCL) membrane. This can be developed further to create a POC device to quantify cell-free DNA, which is a prognostic biomarker for severe sepsis patients.
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Polydimethylsiloxane (PDMS) is a silicone-based synthetic material used in various biomedical applications due to its properties, including transparency, flexibility, permeability to gases, and ease of use. Though PDMS facilitates and assists the fabrication of complicated geometries at micro- and nano-scales, it does not optimally interact with cells for adherence and proliferation. Various strategies have been proposed to render PDMS to enhance cell attachment. The majority of these surface modification techniques have been offered for a static cell culture system. However, dynamic cell culture systems such as organ-on-a-chip devices are demanding platforms that recapitulate a living tissue microenvironment's complexity. In organ-on-a-chip platforms, PDMS surfaces are usually coated by extracellular matrix (ECM) proteins, which occur as a result of a physical and weak bonding between PDMS and ECM proteins, and this binding can be degraded when it is exposed to shear stresses. This work reports static and dynamic coating methods to covalently bind collagen within a PDMS-based microfluidic device using polydopamine (PDA). These coating methods were evaluated using water contact angle measurement and atomic force microscopy (AFM) to optimize coating conditions. The biocompatibility of collagen-coated PDMS devices was assessed by culturing primary human bronchial epithelial cells (HBECs) in microfluidic devices. It was shown that both PDA coating methods could be used to bind collagen, thereby improving cell adhesion (approximately three times higher) without showing any discernible difference in cell attachment between these two methods. These results suggested that such a surface modification can help coat extracellular matrix protein onto PDMS-based microfluidic devices.
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The virusâ»host protein interactions that underlie respiratory syncytial virus (RSV) assembly are still not completely defined, despite almost 60 years of research. RSV buds from the apical surface of infected cells, once virion components have been transported to the budding sites. Association of RSV matrix (M) protein with the actin cytoskeleton may play a role in facilitating this transport. We have investigated the interaction of M with actin in vitro and cell culture. Purified wildtype RSV M protein was found to bind directly to polymerized actin in vitro. Vero cells were transfected to express full-length M (1â»256) as a green fluorescent protein-(GFP) tagged protein, followed by treatment with the microfilament destabilizer, cytochalasin D. Destabilization of the microfilament network resulted in mislocalization of full-length M, from mostly cytoplasmic to diffused across both cytoplasm and nucleus, suggesting that M interacts with microfilaments in this system. Importantly, treatment of RSV-infected cells with cytochalasin D results in lower infectious virus titers, as well as mislocalization of M to the nucleus. Finally, using deletion mutants of M in a transfected cell system, we show that both the N- and C-terminus of the protein are required for the interaction. Together, our data suggest a possible role for Mâ»actin interaction in transporting virion components in the infected cell.
Assuntos
Actinas/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas da Matriz Viral/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citocalasina D/farmacologia , Citoplasma/metabolismo , Deleção de Genes , Humanos , Ligação Proteica/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Vírion/metabolismo , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription.