Microfluidic systems for infectious disease diagnostics.

Microorganisms, encompassing both uni- and multicellular entities, exhibit remarkable diversity as omnipresent life forms in nature. They play a pivotal role by supplying essential components for sustaining biological processes across diverse ecosystems, including higher host organisms. The complex interactions within the human gut microbiota are crucial for metabolic functions, immune responses, and biochemical signalling, particularly through the gut-brain axis. Viruses also play important roles in biological processes, for example by increasing genetic diversity through horizontal gene transfer when replicating inside living cells. On the other hand, infection of the human body by microbiological agents may lead to severe physiological disorders and diseases. Infectious diseases pose a significant burden on global healthcare systems, characterized by substantial variations in the epidemiological landscape. Fast spreading antibiotic resistance or uncontrolled outbreaks of communicable diseases are major challenges at present. Furthermore, delivering field-proven point-of-care diagnostic tools to the most severely affected populations in low-resource settings is particularly important and challenging. New paradigms and technological approaches enabling rapid and informed disease management need to be implemented. In this respect, infectious disease diagnostics taking advantage of microfluidic systems combined with integrated biosensor-based pathogen detection offers a host of innovative and promising solutions. In this review, we aim to outline recent activities and progress in the development of microfluidic diagnostic tools. Our literature research mainly covers the last 5 years. We will follow a classification scheme based on the human body systems primarily involved at the clinical level or on specific pathogen transmission modes. Important diseases, such as tuberculosis and malaria, will be addressed more extensively.

Association between myasthenia gravis and Alzheimer's disease. / Miastenia gravis y enfermedad de Alzheimer: una asociación a estudio.

INTRODUCTION: Myasthenia gravis (MG) and Alzheimer's disease (AD) are two of the most important diseases where the dysregulation of acetylcholine activity plays a crucial role. In the first, this dysregulation happens at the level of the neu-romuscular junction and in the second, in the central nervous system (CNS). AIM: To analyze the possible relationship between these two pathologies, analyzing the prevalence and the odds ratio of AD within patients previously diagnosed with MG. We will compare these data with respect to the prevalence of AD in the general population. PATIENTS AND METHODS: We examined the data obtained by the electronic medical records of patients in the health care system of Castilla La Mancha using the Natural Language Process provided by a clinical platform of artificial intelligence known as the Savana Manager?. RESULTS: We identified 970,503 patients over the age of 60 years, of which 1,028 were diagnosed with MG. The proportion of the patients diagnosed with AD within this group (4.28%) was greater than the rest of the population (2.82%) (p = 0,0047) with an odds ratio of 1.54 (confidence interval at 95% 1.13-2.08; p = 0.0051) without finding significant differences in the bivariate analysis for the rest of the most important actual known risk factors for AD. CONCLUSION: Our results suggest that there might be an increase in the prevalence of AD in patients previously diagnosed with MG.

TITLE: Miastenia gravis y enfermedad de Alzheimer: una asociación a estudio.Introducción. La miastenia gravis (MG) y la enfermedad de Alzheimer (EA) son dos de las enfermedades neurológicas en cuya fisiopatología interviene la acetilcolina en distintos niveles. En la primera, la alteración de este neurotransmisor se produce en la unión neuromuscular, y en la segunda, en el sistema nervioso central. Objetivo. Analizar la posible relación entre dichas patologías estudiando la prevalencia y la odds ratio de la EA dentro de los pacientes diagnosticados de MG con respecto a la prevalencia de EA en la población general. Pacientes y métodos. Se han examinado datos de las historias clínicas electrónicas del sistema de salud de Castilla-La Mancha utilizando el procesamiento de lenguaje natural a través de la plataforma clínica de inteligencia artificial Savana Manager?. Resultados. Se ha identificado a 970.503 pacientes mayores de 60 años, de los que 1.028 tenían diagnóstico de MG. La proporción de pacientes con diagnóstico de EA dentro de este grupo (4,28%) es mayor que en el resto de la población (2,82%; p = 0,0047), con una odds ratio de 1,54 (intervalo de confianza al 95%: 1,13-2,08; p = 0,0051), sin que se encuentren diferencias significativas en el análisis bivariante del resto de los factores de riesgo para EA más importantes conocidos hasta ahora. Conclusiones. Nuestros resultados sugieren que podría existir un aumento de la prevalencia de EA en pacientes con MG.

Quench dynamics of edge states in a finite extended Su-Schrieffer-Heeger system.

We examine the quench dynamics of an extended Su-Schrieffer-Heeger (SSH) model involving long-range hopping that can hold multiple topological phases. Using winding number diagrams to characterize the system's topological phases geometrically, it is shown that there can be multiple winding number transition paths for a quench between two topological phases. The dependence of the quench dynamics is studied in terms of the survival probability of the fermionic edge modes and postquench transport. For two quench paths between two topological regimes with the same initial and final topological phase, the survival probability of edge states is shown to be strongly dependent on the winding number transition path. This dependence is explained using energy band diagrams corresponding to the paths. Following this, the effect of the winding number transition path on transport is investigated. We find that the velocities of maximum transport channels varied along the winding number transition path. This variation depends on the path we choose, i.e., it increases or decreases depending upon the path. An analysis of the coefficient maps, energy spectrum, and spatial structure of the edge states of the final quench Hamiltonian provides an understanding of the path-dependent velocity variation phenomenon.

In silico protein interaction screening uncovers DONSON's role in replication initiation.

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.

Difference in Intestine Content of Caenorhabditis elegans When Fed on Non-Pathogenic or Pathogenic Bacteria.

We investigated the bacterial food digestion and accumulation in wild-type adult Caenorhabditis elegans (C. elegans) worms that have fed on either non-pathogenic RFP-expressing Escherichia coli (E. coli) OP50 or pathogenic-RFP-expressing Pseudomonas aeruginosa (P. aeruginosa) PAO1 during the first 4 days of adulthood. Once the worms had completed their planned feeding cycles, they were loaded on microfluidic chips, where they were fixed to allow high-resolution z-stack fluorescence imaging of their intestines utilizing a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution oil-immersion objective (60×). IMARIS software was used to visualize and analyze the obtained images, resulting in the production of three-dimensional constructs of the intestinal bacterial load. We discovered two distinct patterns for the bacteria-derived fluorescence signal in the intestine: (i) individual fluorescent spots, originating from intact bacteria, were present in the fluorescent E. coli-OP50-fed worms, and (ii) individual fluorescent spots (originating from intact bacteria) were dispersed in large regions of diffuse fluorescence (RDF), originating from disrupted bacteria, in fluorescent P. aeruginosa-PAO1-fed worms. We performed a semi-automated single-worm-resolution quantitative analysis of the intestinal bacterial load, which showed that the intestinal bacterial load generally increases with age of the worms, but more rapidly for the fluorescent P. aeruginosa-PAO1-fed worms.

Acoustofluidic large-scale mixing for enhanced microfluidic immunostaining for tissue diagnostics.

The usage of microfluidics for automated and fast immunoassays has gained a lot of interest in the last decades. This integration comes with certain challenges, like the reconciliation of laminar flow patterns of micro-scale systems with diffusion-limited mass transport. Several methods have been investigated to enhance microfluidic mixing in microsystems, including acoustic-based fluidic streaming. Here, we report both by numerical simulation and experiments on the beneficiary effect of acoustic agitation on the uniformity of immunostaining in large-size and thin microfluidic chambers. Moreover, we investigate by numerical simulation the impact of reducing the incubation times and the concentrations of the biochemical detection reagents on the obtained immunoassay signal. Finally, acoustofluidic mixing was successfully used to reduce by 80% the incubation time of the Her2 (human epidermal growth factor receptor 2) and CK (cytokeratins) biomarkers for the spatial immunostaining of breast cancer cell pellets, or reducing their concentration by 66% and achieving a higher signal-to-background ratio than comparable spatially resolved immunostaining with static incubation.

High-resolution imaging and analysis of the intestinal bacterial load of Caenorhabditis elegans during early adulthood.

We study the presence within the worm Caenorhabditis elegans (C. elegans) of a fluorescent strain of the worm's bacterial food (Escherichia coli (E. coli) OP50) during early adulthood. Use of a microfluidic chip based on a thin glass coverslip substrate allows investigation of the intestinal bacterial load using a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution objective (60×). High-resolution z-stack fluorescence images of the gut bacteria in adult worms, which were loaded in the microfluidic chip and subsequently fixed, were analyzed using IMARIS software and 3D reconstructions of the intestinal bacterial load in the worms were obtained. We present an automated bivariate histogram analysis of the volumes and intensities of the bacterial spots for each worm and find that, as the worms age, the bacterial load in their hindguts increases. We show the advantage of single-worm resolution automated analysis for bacterial load studies and anticipate that the methods described in our work can be easily implemented in existing microfluidic solutions to enable thorough studies of bacterial proliferation.

Host, environment, and anthropogenic factors drive landscape dynamics of an environmentally transmitted pathogen: Sarcoptic mange in the bare-nosed wombat.

Understanding the spatial dynamics and drivers of wildlife pathogens is constrained by sampling logistics, with implications for advancing the field of landscape epidemiology and targeted allocation of management resources. However, visually apparent wildlife diseases, when combined with remote-surveillance and distribution modelling technologies, present an opportunity to overcome this landscape-scale problem. Here, we investigated dynamics and drivers of landscape-scale wildlife disease, using clinical signs of sarcoptic mange (caused by Sarcoptes scabiei) in its bare-nosed wombat (BNW; Vombatus ursinus) host. We used 53,089 camera-trap observations from over 3261 locations across the 68,401 km2 area of Tasmania, Australia, combined with landscape data and ensemble species distribution modelling (SDM). We investigated: (1) landscape variables predicted to drive habitat suitability of the host; (2) host and landscape variables associated with clinical signs of disease in the host; and (3) predicted locations and environmental conditions at greatest risk of disease occurrence, including some Bass Strait islands where BNW translocations are proposed. We showed that the Tasmanian landscape, and ecosystems therein, are nearly ubiquitously suited to BNWs. Only high mean annual precipitation reduced habitat suitability for the host. In contrast, clinical signs of sarcoptic mange disease in BNWs were widespread, but heterogeneously distributed across the landscape. Mange (which is environmentally transmitted in BNWs) was most likely to be observed in areas of increased host habitat suitability, lower annual precipitation, near sources of freshwater and where topographic roughness was minimal (e.g. human modified landscapes, such as farmland and intensive land-use areas, shrub and grass lands). Thus, a confluence of host, environmental and anthropogenic variables appear to influence the risk of environmental transmission of S. scabiei. We identified that the Bass Strait Islands are highly suitable for BNWs and predicted a mix of high and low suitability for the pathogen. This study is the largest spatial assessment of sarcoptic mange in any host species, and advances understanding of the landscape epidemiology of environmentally transmitted S. scabiei. This research illustrates how host-pathogen co-suitability can be useful for allocating management resources in the landscape.

Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle.

Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.

Correction: Bubble-enhanced ultrasonic microfluidic chip for rapid DNA fragmentation.

Correction for 'Bubble-enhanced ultrasonic microfluidic chip for rapid DNA fragmentation' by Lin Sun et al., Lab Chip, 2022, 22, 560-572, https://doi.org/10.1039/D1LC00933H.

Efficient AC electrothermal flow (ACET) on-chip for enhanced immunoassays.

Biochemical reaction rates in microfluidic systems are known to be limited by the diffusional transport of reagents, leading often to lowered sensitivity and/or longer detection times in immunoassays. Several methods, including electrically powering electrodes to generate AC electrothermal flow (ACET) on-chip, have been adopted to enhance the mass transport of the reagents and improve microfluidic mixing. Here, we report a novel ACET electrode design concept for generating in-plane microfluidic mixing vortices that act over a large volume close to the reaction surface of interest. This is different from the traditional ACET parallel electrode design that provides rather local vertical mixing vortices directly above the electrodes. Both numerical simulation and experimental studies were performed to validate the new design. Moreover, numerical simulation was carried out to show the effects of experimental factors such as the reaction kinetics (association constant) and the reagent concentration on the ACET-enhanced surface-based assays. As a proof of concept, the new design for the ACET-enhanced immunoassays was used to improve the immunostaining signal of the HER2 (human epidermal growth factor receptor 2) cancer biomarker on breast cancer cells. Finally, the concept of scaling up the design has been validated by experiments (immunoassays on breast cancer cells for different ACET power and different assay times). In particular, we show that larger ACET in-plane designs can agitate and mix the fluid over large microfluidic volumes, which further enhances the immunoassay's output. We have achieved a 6-times enhancement in the assay signal with a 75% reduction in assay time.

Polydimethylsiloxane microstructure-induced acoustic streaming for enhanced ultrasonic DNA fragmentation on a microfluidic chip.

Next-generation sequencing (NGS) is an essential technology for DNA identification in genomic research. DNA fragmentation is a critical step for NGS and doing this on-chip is of great interest for future integrated genomic solutions. Here we demonstrate fast acoustofluidic DNA fragmentation via ultrasound-actuated elastic polydimethylsiloxane (PDMS) microstructures that induce acoustic streaming and associated shear forces when placed in the field of an ultrasonic transducer. Indeed, acoustic streaming locally generates high tensile stresses that can mechanically stretch and break DNA molecule chains. The improvement in efficiency of the on-chip DNA fragmentation is due to the synergistic effect of these tensile stresses and ultrasonic cavitation phenomena. We tested these microstructure-induced effects in a DNA-containing microfluidic channel both experimentally and by simulation. The DNA fragmentation process was evaluated by measuring the change in the DNA fragment size over time. The chip works well with both long and short DNA chains; in particular, purified lambda (λ) DNA was cut from 48.5 kbp to 3 kbp in one minute with selected microstructures and further down to 300 bp within two and a half minutes. The fragment size of mouse genomic DNA was reduced from 1.4 kbp to 400 bp in one minute and then to 200 bp in two and a half minutes. The DNA fragmentation efficiency of the chip equipped with the PDMS microstructures was twice that of the chip without the microstructures. Exhaustive comparison shows that the on-chip fragmentation performance reaches the level of high-end professional standards. Recently, DNA fragmentation was shown to be enhanced using vibrating air microbubbles when the chip was placed in an acoustic field. We think the microbubble-free microstructure-based device we present is easier to operate and more reliable, as it avoids microbubble preparation and maintenance, while showing high DNA fragmentation performance.

The enhancement of DNA fragmentation in a bench top ultrasonic water bath with needle-induced air bubbles: Simulation and experimental investigation.

Shearing DNA to a certain size is the first step in many medical and biological applications, especially in next-generation gene sequencing technology. In this article, we introduced a highly efficient ultrasonic DNA fragmentation method enhanced by needle-induced air bubbles, which is easy to operate with high throughput. The principle of the bubble-enhanced sonication system is introduced and verified by flow field and acoustic simulations and experiments. Lambda DNA long chains and mouse genomic DNA short chains are used in the experiments for testing the performance of the bubble-enhanced ultrasonic DNA fragmentation system. Air bubbles are an effective enhancement agent for ultrasonic DNA fragmentation; they can obviously improve the sound pressure level in the whole solution, thus, achieving better absorption of ultrasound energy. Growing bubbles also have a stretched function on DNA molecule chains and form a huge pressure gradient in the solution, which is beneficial to DNA fragmentation. Purified λDNA is cut from 48.5 to 2 kbp in 5 min and cut to 300 bp in 30 min. Mouse genomic DNA (≈1400 bp) decreases to 400 bp in 5 min and then reduces to 200 bp in 30 min. This bubble-enhanced ultrasonic method enables widespread access to genomic DNA fragmentation in a standard ultrasonic water bath for many virus sequencing demands even without good medical facilities.

User acceptability of saliva and gargle samples for identifying COVID-19 positive high-risk workers and household contacts.

Throughout the COVID-19 pandemic nasopharyngeal or nose and/or throat swabs (NTS) have been the primary approach for collecting patient samples for the subsequent detection of viral RNA. However, this procedure, if undertaken correctly, can be unpleasant and therefore deters individuals from providing high quality samples. To overcome these limitations other modes of sample collection have been explored. In a cohort of frontline health care workers we have compared saliva and gargle samples to gold-standard NTS. 93% of individuals preferred providing saliva or gargle samples, with little sex-dependent variation. Viral titers collected in samples were analyzed using standard methods and showed that gargle and saliva were similarly comparable for identifying COVID-19 positive individuals compared to NTS (92% sensitivity; 98% specificity). We suggest that gargle and saliva collection are viable alternatives to NTS swabs and may encourage testing to provide better disease diagnosis and population surveillance.

DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières syndrome astrocytes.

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

Reconciling communication repertoires: navigating interactions involving persons with severe/profound intellectual disability, a classic grounded theory study.

BACKGROUND: A rights-based agenda, informed by the UNCRPD, that advocates person-centredness, inclusion, empowerment and self-determination is shaping service provision to people with intellectual disability (ID). Listening to their perspectives is fundamental to meeting these goals. However, communication with people with severe/profound ID is challenging and difficult. Therefore, this study aims to generate a theory that explains how people communicate with and understand each other in these interactions. METHODS: Classic grounded theory (CGT) methodology was used as it recognises that knowledge can be captured rather than interpreted. According to CGT, capturing rather than interpreting experiences strengthens findings, particularly in relation to participants with severe/profound ID. Concurrent theoretical sampling, data collection and analysis were undertaken. Twenty-two individuals participated in the study: 3 people with severe/profound ID and 19 people with whom they interact. Data were collected over a 9-month period and involved video recordings, field notes, individual and group interviews. Data were analysed using CGT methods of coding, constant comparison and memoing. RESULTS: The Theory of Reconciling Communication Repertoires was generated. Nurturing a sense of belonging emerged as the main concern and core category that is resolved by reconciling communication repertoires. A communication repertoire refers to the cache of communication skills a person has available to them. To reconcile repertoires is to harmonise or make them compatible with each other in order to communicate. Interactions are navigated through five stages: motivation to interact, connection establishment, reciprocally engaging, navigating understanding and confusion resolution. CONCLUSIONS: The Theory of Reconciling Communication Repertoires explains how interactions involving people with severe/profound ID are navigated. While this is a substantive rather than formal theory, it has the potential to inform practice, policy, management, education and research as it outlines how communication with people with severe/profound ID can take place to design, inform and plan person-centred care.