A Lab-in-a-Fiber optofluidic device using droplet microfluidics and laser-induced fluorescence for virus detection.

Microfluidics has emerged rapidly over the past 20 years and has been investigated for a variety of applications from life sciences to environmental monitoring. Although continuous-flow microfluidics is ubiquitous, segmented-flow or droplet microfluidics offers several attractive features. Droplets can be independently manipulated and analyzed with very high throughput. Typically, microfluidics is carried out within planar networks of microchannels, namely, microfluidic chips. We propose that fibers offer an interesting alternative format with key advantages for enhanced optical coupling. Herein, we demonstrate the generation of monodisperse droplets within a uniaxial optofluidic Lab-in-a-Fiber scheme. We combine droplet microfluidics with laser-induced fluorescence (LIF) detection achieved through the development of an optical side-coupling fiber, which we term a periscope fiber. This arrangement provides stable and compact alignment. Laser-induced fluorescence offers high sensitivity and low detection limits with a rapid response time making it an attractive detection method for in situ real-time measurements. We use the well-established fluorophore, fluorescein, to characterize the Lab-in-a-Fiber device and determine the generation of [Formula: see text] 0.9 nL droplets. We present characterization data of a range of fluorescein concentrations, establishing a limit of detection (LOD) of 10 nM fluorescein. Finally, we show that the device operates within a realistic and relevant fluorescence regime by detecting reverse-transcription loop-mediated isothermal amplification (RT-LAMP) products in the context of COVID-19 diagnostics. The device represents a step towards the development of a point-of-care droplet digital RT-LAMP platform.

Molecular detection of Gram-positive bacteria in the human lung through an optical fiber-based endoscope.

PURPOSE: The relentless rise in antimicrobial resistance is a major societal challenge and requires, as part of its solution, a better understanding of bacterial colonization and infection. To facilitate this, we developed a highly efficient no-wash red optical molecular imaging agent that enables the rapid, selective, and specific visualization of Gram-positive bacteria through a bespoke optical fiber-based delivery/imaging endoscopic device. METHODS: We rationally designed a no-wash, red, Gram-positive-specific molecular imaging agent (Merocy-Van) based on vancomycin and an environmental merocyanine dye. We demonstrated the specificity and utility of the imaging agent in escalating in vitro and ex vivo whole human lung models (n = 3), utilizing a bespoke fiber-based delivery and imaging device, coupled to a wide-field, two-color endomicroscopy system. RESULTS: The imaging agent (Merocy-Van) was specific to Gram-positive bacteria and enabled no-wash imaging of S. aureus within the alveolar space of whole ex vivo human lungs within 60 s of delivery into the field-of-view, using the novel imaging/delivery endomicroscopy device. CONCLUSION: This platform enables the rapid and specific detection of Gram-positive bacteria in the human lung.

Core crosstalk in ordered imaging fiber bundles.

Coherent fiber bundles are used widely for imaging. Commonly, disordered arrays of randomly sized fiber cores avoid proximity between like-cores, which would otherwise result in increased core crosstalk and a negative impact on imaging. Recently, stack-and-draw fiber manufacture techniques have been used to produce fibers with a controlled core layout to minimize core crosstalk. However, one must take manufacturing considerations into account during stack-and-draw fiber design in order to avoid impractical or unachievable fabrication. This comes with a set of practical compromises, such as using only a small number of different core sizes. Through characterization of core crosstalk patterns, this Letter aims to aid the understanding of crosstalk limitations imposed by such compromises in the core layout made for ease of fabrication.

Frugal filtering optical lenses for point-of-care diagnostics.

Infectious diseases are the leading cause of morbidity and mortality in low and middle income countries (LMICs). Rapid diagnosis of infections in LMICs presents many challenges, especially in rural areas where access to health care, including diagnostics, is poor. Microscopy is one of the most commonly used platforms to diagnose bacterial infections on clinical samples. Fluorescence microscopy has high sensitivity and specificity but to date is mostly performed within a laboratory setting due to the high-cost, low portability and highly specialist nature of equipment. Point-of-care diagnostics could offer a solution to the challenge of infection diagnosis in LMICs. In this paper we present frugal, easy to manufacture, doped polydimethylsiloxane filtering optical lenses that can be integrated into smartphone microscopes for immediate detection of fluorescently labelled bacteria. This provides a breakthrough technology platform for point-of-care diagnostics.

Demonstrating the Use of Optical Fibres in Biomedical Sensing: A Collaborative Approach for Engagement and Education.

This paper demonstrates how research at the intersection of physics, engineering, biology and medicine can be presented in an interactive and educational way to a non-scientific audience. Interdisciplinary research with a focus on prevalent diseases provides a relatable context that can be used to engage with the public. Respiratory diseases are significant contributors to avoidable morbidity and mortality and have a growing social and economic impact. With the aim of improving lung disease understanding, new techniques in fibre-based optical endomicroscopy have been recently developed. Here, we present a novel engagement activity that resembles a bench-to-bedside pathway. The activity comprises an inexpensive educational tool (<$70) adapted from a clinical optical endomicroscopy system and tutorials that cover state-of-the-art research. The activity was co-created by high school science teachers and researchers in a collaborative way that can be implemented into any engagement development process.

Fibre-based spectral ratio endomicroscopy for contrast enhancement of bacterial imaging and pulmonary autofluorescence.

Fibre-based optical endomicroscopy (OEM) permits high resolution fluorescence microscopy in endoscopically accessible tissues. Fibred OEM has the potential to visualise pathologies targeted with fluorescent imaging probes and provide an in vivo in situ molecular pathology platform to augment disease understanding, diagnosis and stratification. Here we present an inexpensive widefield ratiometric fibred OEM system capable of enhancing the contrast between similar spectra of pathologically relevant fluorescent signals without the burden of complex spectral unmixing. As an exemplar, we demonstrate the potential of the platform to detect fluorescently labelled Gram-negative bacteria in the challenging environment of highly autofluorescent lung tissue in whole ex vivo human lungs.

Characterization and modelling of inter-core coupling in coherent fiber bundles.

Recent developments in optical endomicroscopy (OEM) and associated fluorescent SmartProbes present a need for sensitive imaging with high detection performance. Inter-core coupling within coherent fiber bundles is a well recognized limitation, affecting the technology's imaging capabilities. Fiber cross coupling has been studied both experimentally and within a theoretical framework (coupled mode theory), providing (i) insights on the factors affecting cross talk, and (ii) recommendations for optimal fiber bundle design. However, due to physical limitations, such as the tradeoff between cross coupling and core density, cross coupling can be suppressed yet not eliminated through optimal fiber design. This study introduces a novel approach for measuring, analyzing and quantifying cross coupling within coherent fiber bundles, in a format that can be integrated into a linear model, which in turn can enable computational compensation of the associated blurring introduced to OEM images.

The role of gut endocrine cells in control of metabolism and appetite.

After food is ingested, nutrients pass through the gastrointestinal tract, stimulating the release of a range of peptide hormones. Among their many local, central and peripheral actions, these hormones act to mediate glucose metabolism and satiety. Indeed, it is the modification of gut hormone secretion that is considered partly responsible for the normalization of glycaemic control and the reduction in appetite seen in many patients after certain forms of bariatric surgery. This review describes recent developments in our understanding of the secretion and action of anorexigenic gut hormones, primarily concentrating on glucagon-like peptide-1 (GLP-1).

Identification and characterization of GLP-1 receptor-expressing cells using a new transgenic mouse model.

GLP-1 is an intestinal hormone with widespread actions on metabolism. Therapies based on GLP-1 are highly effective because they increase glucose-dependent insulin secretion in people with type 2 diabetes, but many reports suggest that GLP-1 has additional beneficial or, in some cases, potentially dangerous actions on other tissues, including the heart, vasculature, exocrine pancreas, liver, and central nervous system. Identifying which tissues express the GLP-1 receptor (GLP1R) is critical for the development of GLP-1-based therapies. Our objective was to use a method independent of GLP1R antibodies to identify and characterize the targets of GLP-1 in mice. Using newly generated glp1r-Cre mice crossed with fluorescent reporter strains, we show that major sites of glp1r expression include pancreatic ß- and δ-cells, vascular smooth muscle, cardiac atrium, gastric antrum/pylorus, enteric neurones, and vagal and dorsal root ganglia. In the central nervous system, glp1r-fluorescent cells were abundant in the area postrema, arcuate nucleus, paraventricular nucleus, and ventromedial hypothalamus. Sporadic glp1r-fluorescent cells were found in pancreatic ducts. No glp1r-fluorescence was observed in ventricular cardiomyocytes. Enteric and vagal neurons positive for glp1r were activated by GLP-1 and may contribute to intestinal and central responses to locally released GLP-1, such as regulation of intestinal secretomotor activity and appetite.

Oligopeptides stimulate glucagon-like peptide-1 secretion in mice through proton-coupled uptake and the calcium-sensing receptor.

AIMS/HYPOTHESIS: Ingested protein is a well-recognised stimulus for glucagon-like peptide-1 (GLP-1) release from intestinal L cells. This study aimed to characterise the molecular mechanisms employed by L cells to detect oligopeptides. METHODS: GLP-1 secretion from murine primary colonic cultures and Ca(2+) dynamics in L cells were monitored in response to peptones and dipeptides. L cells were identified and purified based on their cell-specific expression of the fluorescent protein Venus, using GLU-Venus transgenic mice. Pharmacological tools and knockout mice were used to characterise candidate sensory pathways identified by expression analysis. RESULTS: GLP-1 secretion was triggered by peptones and di-/tripeptides, including the non-metabolisable glycine-sarcosine (Gly-Sar). Two sensory mechanisms involving peptide transporter-1 (PEPT1) and the calcium-sensing receptor (CaSR) were distinguishable. Responses to Gly-Sar (10 mmol/l) were abolished in the absence of extracellular Ca(2+) or by the L-type calcium-channel blocker nifedipine (10 µmol/l) and were PEPT1-dependent, as demonstrated by their sensitivity to pH and 4-aminomethylbenzoic acid and the finding of impaired responses in tissue from Pept1 (also known as Slc15a1) knockout mice. Peptone (5 mg/ml)-stimulated Ca(2+) responses were insensitive to nifedipine but were blocked by antagonists of CaSR. Peptone-stimulated GLP-1 secretion was not impaired in mice lacking the putative peptide-responsive receptor lysophosphatidic acid receptor 5 (LPAR5; also known as GPR92/93). CONCLUSIONS/INTERPRETATION: Oligopeptides stimulate GLP-1 secretion through PEPT1-dependent electrogenic uptake and activation of CaSR. Both pathways are highly expressed in native L cells, and likely contribute to the ability of ingested protein to elevate plasma GLP-1 levels. Targeting nutrient-sensing pathways in L cells could be used to mobilise endogenous GLP-1 stores in humans, and could mimic some of the metabolic benefits of bariatric surgery.

Overlap of endocrine hormone expression in the mouse intestine revealed by transcriptional profiling and flow cytometry.

The intestine secretes a range of hormones with important local and distant actions, including the control of insulin secretion and appetite. A number of enteroendocrine cell types have been described, each characterized by a distinct hormonal signature, such as K-cells producing glucose-dependent insulinotropic polypeptide (GIP), L-cells producing glucagon-like peptide-1 (GLP-1), and I-cells producing cholecystokinin (CCK). To evaluate similarities between L-, K-, and other enteroendocrine cells, primary murine L- and K-cells, and pancreatic α- and ß-cells, were purified and analyzed by flow cytometry and microarray-based transcriptomics. By microarray expression profiling, L cells from the upper small intestinal (SI) more closely resembled upper SI K-cells than colonic L-cells. Upper SI L-cell populations expressed message for hormones classically localized to different enteroendocrine cell types, including GIP, CCK, secretin, and neurotensin. By immunostaining and fluorescence-activated cell sorting analysis, most colonic L-cells contained GLP-1 and PeptideYY In the upper SI, most L-cells contained CCK, approximately 10% were GIP positive, and about 20% were PeptideYY positive. Upper SI K-cells exhibited approximately 10% overlap with GLP-1 and 6% overlap with somatostatin. Enteroendocrine-specific transcription factors were identified from the microarrays, of which very few differed between the enteroendocrine cell populations. Etv1, Prox1, and Pax4 were significantly enriched in L-cells vs. K cells by quantitative RT-PCR. In summary, our data indicate a strong overlap between upper SI L-, K-, and I-cells and suggest they may rather comprise a single cell type, within which individual cells exhibit a hormonal spectrum that may reflect factors such as location along the intestine and exposure to dietary nutrients.

Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion.

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.

Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.

The associations between physical activity, screen time and weight from 6 to 14 yrs: the Raine Study.

To examine the strength and direction of the relationship between physical activity level, screen use and BMI in a cohort at ages 6, 8, 10 and 14 yrs as part of a prospective longitudinal cohort study. The sample comprised 1403 males and females who participated in the follow-up survey at 14 yrs of age between 2003 and 2005. Exploratory structural equation modelling was used to examine the interrelationships between physical activity level, BMI and screen time at 6, 8, 10 and 14 yrs. Predictors of BMI at 6, 8, 10 and 14 yrs explained 1.3, 76.1, 80.1 and 73.1 percent of the variances, respectively, with previous BMI the largest predictor [χ(2)=43.082, df=36, p=194]. Increased screen time predicted higher BMI and lower physical activity at 8 and 10 yrs but not 14 yrs. At 14 yrs, physical activity predicted BMI. Sedentary patterns of behaviour in early childhood were predictive of later and concurrent obesity, whereas physical activity was predictive of obesity in adolescence. Different intervention targets are required for children and adolescents.

Glutamine triggers and potentiates glucagon-like peptide-1 secretion by raising cytosolic Ca2+ and cAMP.

L-glutamine stimulates glucagon-like peptide 1 (GLP-1) secretion in human subjects and cell lines. As recent advances have enabled the study of primary GLP-1-releasing L cells, this study aimed to characterize glutamine-sensing pathways in native murine L cells. L cells were identified using transgenic mice with cell-specific expression of fluorescent markers. Cells were studied in primary colonic cultures from adult mice, or purified by flow cytometry for expression analysis. Intracellular Ca(2+) was monitored in cultures loaded with Fura2, and cAMP was studied using Förster resonance energy transfer sensors expressed in GLUTag cells. Asparagine, phenylalanine, and glutamine (10 mm) triggered GLP-1 release from primary cultures, but glutamine was the most efficacious, increasing secretion 1.9-fold with an EC(50) of 0.19 mm. Several amino acids triggered Ca(2+) changes in L cells, comparable in magnitude to that induced by glutamine. Glutamine-induced Ca(2+) responses were abolished in low Na(+) solution and attenuated in Ca(2+) free solution, suggesting a role for Na(+) dependent uptake and Ca(2+) influx. The greater effectiveness of glutamine as a secretagogue was paralleled by its ability to increase cAMP in GLUTag cells. Glutamine elevated intracellular cAMP to 36% of that produced by a maximal stimulus, whereas asparagine only increased intracellular cAMP by 24% and phenylalanine was without effect. Glutamine elevates both cytosolic Ca(2+) and cAMP in L cells, which may account for the effectiveness of glutamine as a GLP-1 secretagogue. Therapeutic agents like glutamine that target synergistic pathways in L cells might play a future role in the treatment of type 2 diabetes.

Role of phosphodiesterase and adenylate cyclase isozymes in murine colonic glucagon-like peptide 1 secreting cells.

BACKGROUND AND PURPOSE: Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine L-cells after food intake. Increasing GLP-1 signalling either through inhibition of the GLP-1 degrading enzyme dipeptidyl-peptidase IV or injection of GLP-1-mimetics has recently been successfully introduced for the treatment of type 2 diabetes. Boosting secretion from the L-cell has so far not been exploited, due to our incomplete understanding of L-cell physiology. Elevation of cyclic adenosine monophosphate (cAMP) has been shown to be a strong stimulus for GLP-1 secretion and here we investigate the activities of adenylate cyclase (AC) and phosphodiesterase (PDE) isozymes likely to shape cAMP responses in L-cells. EXPERIMENTAL APPROACH: Expression of AC and PDE isoforms was quantified by RT-PCR. Single cell responses to stimulation or inhibition of AC and PDE isoforms were monitored with real-time cAMP probes. GLP-1 secretion was assessed by elisa. KEY RESULTS: Quantitative PCR identified expression of protein kinase C- and Ca²+-activated ACs, corresponding with phorbolester and cytosolic Ca²+-stimulated cAMP elevation. Inhibition of PDE2, 3 and 4 were found to stimulate GLP-1 secretion from murine L-cells in primary culture. This corresponded with cAMP elevations monitored with a plasma membrane targeted cAMP probe. Inhibition of PDE3 but not PDE2 was further shown to prevent GLP-1 secretion in response to guanylin, a peptide secreted into the gut lumen, which had not previously been implicated in L-cell secretion. CONCLUSIONS AND IMPLICATIONS: Our results reveal several mechanisms shaping cAMP responses in GLP-1 secreting cells, with some of the molecular components specifically expressed in L-cells when compared with their epithelial neighbours, thus opening new strategies for targeting these cells therapeutically.

Molecular mechanisms underlying nutrient-stimulated incretin secretion.

The incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are released from enteroendocrine cells in the intestinal epithelium in response to nutrient ingestion. The actions of GLP-1 and GIP - not only on local gut physiology but also on glucose homeostasis, appetite control and fat metabolism - have made these hormones an attractive area for drug discovery programmes. The potential range of strategies to target the secretion of these hormones therapeutically has been limited by an incomplete understanding of the mechanisms underlying their release. The use of organ and whole-animal perfusion techniques, cell line models and primary L- and K-cells has led to the identification of a variety of pathways involved in the sensing of carbohydrate, fat and protein in the gut lumen. This review focuses on our current understanding of these signalling mechanisms that might underlie nutrient responsiveness of L- and K-cells.

Glucose sensing in L cells: a primary cell study.

Glucagon-like peptide-1 (GLP-1) is an enteric hormone that stimulates insulin secretion and improves glycaemia in type 2 diabetes. Although GLP-1-based treatments are clinically available, alternative strategies to increase endogenous GLP-1 release from L cells are hampered by our limited physiological understanding of this cell type. By generating transgenic mice with L cell-specific expression of a fluorescent protein, we studied the characteristics of primary L cells by electrophysiology, fluorescence calcium imaging, and expression analysis and show that single L cells are electrically excitable and glucose responsive. Sensitivity to tolbutamide and low-millimolar concentrations of glucose and alpha-methylglucopyranoside, assessed in single L cells and by hormone secretion from primary cultures, suggested that GLP-1 release is regulated by the activity of sodium glucose cotransporter 1 and ATP-sensitive K(+) channels, consistent with their high expression levels in purified L cells by quantitative RT-PCR. These and other pathways identified using this approach will provide exciting opportunities for future physiological and therapeutic exploration.