Comparison between capillary electrophoresis and fluorescence anisotropy competitive immunoassay for glucagon.

Glucagon plays a crucial role in regulating glucose homeostasis; unfortunately, the mechanisms controlling its release are still unclear. Capillary electrophoresis (CE)- and fluorescence anisotropy (FA)-immunoassays (IA) have been used for online measurements of hormone secretion on microfluidic platforms, although their use in glucagon assays is less common. We set out to compare a glucagon-competitive IA using these two techniques. Theoretical calibration curves were generated for both CE- and FA-IA and results indicated that CE-IA provided higher sensitivity than FA-IA. These results were confirmed in an experiment where both assays showed limits of detection (LOD) of 30 nM, but the CE-IA had ∼300-fold larger sensitivity from 0 to 200 nM glucagon. However, in online experiments where reagents were mixed within the device, the sensitivity of the CE-IA was reduced ∼3-fold resulting in a higher LOD of 70 nM, whereas the FA-IA remained essentially unchanged. This lowered sensitivity in the online CE-IA was likely due to poor sampling by electroosmotic flow from the high salt solution necessary in online experiments, whereas pressure-based sampling used in FA-IA was not affected. We conclude that FA-IA, despite lowered sensitivity, is more suitable for online mixing scenarios due to the ability to use pressure-driven flow and other practical advantages such as the use of larger channels.

Relations between Glucose and d-Amino Acids in the Modulation of Biochemical and Functional Properties of Rodent Islets of Langerhans.

The cell-to-cell signaling role of d-amino acids (d-AAs) in the mammalian endocrine system, particularly in the islets of Langerhans, has drawn growing interest for their potential involvement in modulating glucose metabolism. Previous studies found colocalization of serine racemase [produces d-serine (d-Ser)] and d-alanine (d-Ala) within insulin-secreting beta cells and d-aspartate (d-Asp) within glucagon-secreting alpha cells. Expressed in the islets, functional N-methyl-d-aspartate receptors are involved in the modulation of glucose-stimulated insulin secretion and have binding sites for several d-AAs. However, knowledge of the regulation of d-AA levels in the islets during glucose stimulation as well as the response of islets to different levels of extracellular d-AAs is limited. In this study, we determined the intracellular and extracellular levels of d-Ser, d-Ala, and d-Asp in cultures of isolated rodent islets exposed to different levels of extracellular glucose. We found that the intracellular levels of the enantiomers demonstrated large variability and, in general, were not affected by extracellular glucose levels. However, significantly lower levels of extracellular d-Ser and d-Ala were observed in the islet media supplemented with 20 mM concentration of glucose compared to the control condition utilizing 3 mM glucose. Glucose-induced oscillations of intracellular free calcium concentration ([Ca2+]i), a proxy for insulin secretion, were modulated by the exogenous application of d-Ser and d-Ala but not by their l-stereoisomers. Our results provide new insights into the roles of d-AAs in the biochemistry and function of pancreatic islets.

Analysis of D-amino acids secreted from murine islets of Langerhans using Marfey's reagent and reversed phase LC-MS/MS.

D-amino acids (D-AAs) are important signaling molecules due to their ability to bind ionotropic N-methyl-D-aspartate receptors. D-serine (D-Ser), D-alanine (D-Ala), and D-aspartate (D-Asp) have been found individually in the endocrine portion of the pancreas, the islets of Langerhans, and/or their secretions. However, there has been no report of a comprehensive assessment of D-AAs in islet secretions. To evaluate the release of these compounds, the effectiveness of both 1-(9-fluorenyl)-ethyl chloroformate (FLEC reagent) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent, MR) in separation of D/L-AA enantiomeric pairs in islet-specific buffers were evaluated. MR-derivatized D/L AAs showed greater than baseline resolution (Rs ≥ 1.5) of 13 enantiomeric pairs when using a non-linear gradient and an acidic mobile phase system, while FLEC-derivatized AAs exhibited limited resolution on both biphenyl and C18 columns. The optimized MR method yielded highly reproducible separations with retention times less than 1% RSD. Excellent linearity between the analyte concentrations and response (R2 > 0.98) were obtained, with less than 15% RSD for all analyte responses. Most analytes had an LOD at or below 100 nM, except for L-Ala (200 nM). The optimized MR method was used to quantify D-AAs in secretions of 150 murine islets after incubation in 3- and 20-mM glucose. In response to both solutions, D-Ser and D-glutamine were tentatively identified via comparison of retention time and quantifier-to-qualifer ion ratios with standards, and from spiking experiments. Both were secreted in low quantities which did not differ significantly in either low (D-Ser: 44 ± 2 fmol islet-1h-1; D-Gln: 300 ± 100 fmol islet-1h-1) or high (D-Ser: 23 ± 1 fmol islet-1h-1; D-Gln: 120 ± 50 fmol islet-1h-1) glucose across 3 biological replicates. The method developed is robust and can be applied to further examine the release of D-AAs and their potential roles in islet physiology.

Simultaneous monitoring of multiple hormones from human islets of Langerhans using solid-phase extraction-mass spectrometry.

Islets of Langerhans release peptide hormones in controlled amounts and patterns to ensure proper maintenance of blood glucose levels. The overall release of the hormones is shaped by external factors and by autocrine and paracrine interactions occurring within the islets. To better understand what controls the secretion of islet-secreted peptides, and how these processes go awry in diabetes, methods to monitor the release of multiple hormones simultaneously are needed. While antibody-based assays are typically used, they are most often applied to quantification of a single hormone. Mass spectrometry (MS), on the other hand, is well suited for quantifying multiple hormones simultaneously but typically requires time-consuming separation steps with biological samples. In this report, response surface methodology was used to identify a set of optimal solid-phase extraction (SPE) conditions for the islet-secreted peptides, insulin, C-peptide, glucagon, and somatostatin. The optimized SPE method was used with multiple reaction monitoring and isotopically labeled standards to quantify secretion levels. Calibrations were linear from 0.5 to 50 nM with < 15% RSD peak area ratios. A microfluidic system was used to perfuse 30 human islets with different glucose conditions, and fractions were collected every 2 min for SPE-MS analysis. Results showed the release dynamics of the individual peptides, as well as patterns, such as positively and negatively correlated release and oscillations. This rapid SPE-MS method is expected to be useful for examining other peptide and small-molecule secretions from islets and could be applied to a number of other biological systems for investigating cellular communication.

Automated cellular stimulation with integrated pneumatic valves and fluidic capacitors.

Microfluidic technologies have proven to be a reliable tool in profiling dynamic insulin secretion from islets of Langerhans. Most of these systems rely on external pressure sources to induce flow, leading to difficulties moving to more elaborate systems. To reduce complexity, a microfluidic system was developed that used a single vacuum source at the outlet to drive fluidic transport of immunoassay reagents and stimulation solutions throughout the device. A downside to this approach is the lack of flow control over the reagents delivered to the islet chamber. To address this challenge, 4-layer pneumatic valves were integrated into the perfusion lines to automate and control the delivery of stimulants; however, it was found that as the valves closed, spikes in the flow would lead to abnormal insulin secretion profiles. Fluidic capacitors were then incorporated after the valves and found to remove the spikes. The combination of the valves and capacitors resulted in automated collection of insulin secretion profiles from single murine islets that were similar to those previously reported in the literature. In the future, these integrated fluidic components may enable more complex channel designs to be used with a relatively simple flow control solution.

Increasing insulin measurement throughput by fluorescence anisotropy imaging immunoassays.

Insulin secreted from islets of Langerhans is the main hormone to reduce blood glucose. Examination of insulin secretion patterns at the single islet level reveals functional differences in the timings and patterns of release. This heterogeneous response highlights the importance of developing systems to measure dynamic release from small numbers of islets in parallel. Toward this, we describe fluorescence anisotropy imaging immunoassays as a relatively simple method for increased throughput of islet secretion measurements. In this system, vacuum pressure from a syringe pump pulled perfusate from 12 islet chambers and reagents into 12 parallel mixing channels for a competitive immunoassay. Light from a Xe arc lamp was filtered and polarized prior to focusing on the microfluidic device at the region where the 12 mixing channels converged. Emission was collected and passed through vertical and horizontal emission polarizers housed in an automated filter wheel before being imaged with a sCMOS camera for the determination of anisotropy. This microfluidic system was tested by monitoring insulin release from groups of murine and human islets. Heterogeneity was observed in the islet traces; however, the presence of islets affected the resistance of the islet chambers, hampering insulin quantification. Nonetheless, this microfluidic system is a step towards increasing the throughput of hormone release measurements from islets of Langerhans.

Small molecules released from islets of Langerhans determined by liquid chromatography - mass spectrometry.

Islets of Langerhans are the endocrine tissue within the pancreas that secrete hormones for maintenance of blood glucose homeostasis. A variety of small molecules including classical neurotransmitters are also released from islets. While the roles of most of these small molecules are unknown, some have been hypothesized to play a critical role in islet physiology. To better understand their role on islet function, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify 39 small molecules released from islets. Benzoyl chloride derivatization of analyte molecules was used to impart retention and facilitate electrospray ionization efficiency. Separation was achieved on a 2.1 × 150 mm column packed with 2.7 µm core-shell C18 particles. Calibration curves showed excellent linearity between the concentration and analyte response, with relative standard deviations of the analyte responses below 15% and limits of detection from 0.01-40 nM. The method was applied to examine small molecules released from murine and human islets of Langerhans after static incubation and perfusion with glucose. Results showed a decrease in secretion rates with increasing glucose concentration for most of the analytes. Secretion rates were found to be higher in human islets compared to their murine counterpart. This method will be useful in understanding the roles of small molecules in biological systems.

Finding a Needle in a Haystack - In Silico Search for Environmental Traces of Candida auris.

Candida auris, first described from an ear infection in Japan, is an important emerging multidrug-resistant pathogenic fungal species. Its environmental niche remained a mystery until its isolation from the wetlands of the Andaman Islands, India, in 2020. We screened a subset of the world's largest sequence repository, the Sequence Read Archive at National Center for Biotechnology Information, using a DNA metabarcoding approach based on either the internal transcribed spacer (ITS)1 or ITS2 region of the official primary fungal DNA barcode, to identify potential environmental sources of C. auris. Our search identified 34 matches with partial C. auris ITS sequences from seven metabarcoding studies, providing wider evidence for the presence of C. auris outside human-maintained facilities.

In depth search of the Sequence Read Archive database reveals global distribution of the emerging pathogenic fungus Scedosporium aurantiacum.

Scedosporium species are emerging opportunistic fungal pathogens causing various infections mainly in immunocompromised patients, but also in immunocompetent individuals, following traumatic injuries. Clinical manifestations range from local infections, such as subcutaneous mycetoma or bone and joint infections, to pulmonary colonization and severe disseminated diseases. They are commonly found in soil and other environmental sources. To date S. aurantiacum has been reported only from a handful of countries. To identify the worldwide distribution of this species we screened publicly available sequencing data from fungal metabarcoding studies in the Sequence Read Archive (SRA) of The National Centre for Biotechnology Information (NCBI) by multiple BLAST searches. S. aurantiacum was found in 26 countries and two islands, throughout every climatic region. This distribution is like that of other Scedosporium species. Several new environmental sources of S. aurantiacum including human and bovine milk, chicken and canine gut, freshwater, and feces of the giant white-tailed rat (Uromys caudimaculatus) were identified. This study demonstrated that raw sequence data stored in the SRA database can be repurposed using a big data analysis approach to answer biological questions of interest. LAY SUMMARY: To understand the distribution and natural habitat of S. aurantiacum, species-specific DNA sequences were searched in the SRA database. Our large-scale data analysis illustrates that S. aurantiacum is more widely distributed than previously thought and new environmental sources were identified.

The High Energy Density Scientific Instrument at the European XFEL.

The European XFEL delivers up to 27000 intense (>1012 photons) pulses per second, of ultrashort (≤50 fs) and transversely coherent X-ray radiation, at a maximum repetition rate of 4.5 MHz. Its unique X-ray beam parameters enable groundbreaking experiments in matter at extreme conditions at the High Energy Density (HED) scientific instrument. The performance of the HED instrument during its first two years of operation, its scientific remit, as well as ongoing installations towards full operation are presented. Scientific goals of HED include the investigation of extreme states of matter created by intense laser pulses, diamond anvil cells, or pulsed magnets, and ultrafast X-ray methods that allow their diagnosis using self-amplified spontaneous emission between 5 and 25 keV, coupled with X-ray monochromators and optional seeded beam operation. The HED instrument provides two target chambers, X-ray spectrometers for emission and scattering, X-ray detectors, and a timing tool to correct for residual timing jitter between laser and X-ray pulses.

A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

Glucagon is a 29-amino acid peptide released from α-cells within pancreatic islets of Langerhans to help raise blood glucose levels. While a plethora of methodologies have been developed for quantitative measurement of insulin released from islets, such methods are not well developed for glucagon despite its importance in blood sugar regulation. In this work, a simple yet robust microfluidic device was developed for holding human pancreatic islets and perfuse them with glucose. The perfusate was collected into 2 min fractions and glucagon quantified using a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay. Simulation of fluid flow within the microfluidic device indicated the device produced low amounts of shear stress on islets, and characterization of the flow with standard glucagon solutions revealed response times within 2 fractions (<4 min). Results with human islets from multiple donors demonstrated either a "burst" of glucagon or a "sustained" glucagon release across the entire period of stimulation. The simplicity, yet robustness, of the device and method is expected to appeal to a number of researchers examining pancreatic islet physiology.

Effect of deglycosylation on immunoreactivity and in vitro pepsin digestibility of major cashew (Anacardium occidentale L.) allergen, Ana o 1.

Major cashew allergen, Ana o 1, was purified in its native form from cashew seeds and subjected to enzymatic deglycosylation using PNGase F to assess the potential role of N-glycans in immunoreactivity. Western and dot blotting with pooled human plasma containing anticashew IgE revealed that deglycosylation increased IgE-binding of Ana o 1. Removal of N-glycans may have exposed previously masked Ana o 1 epitopes. Purified glycosylated and deglycosylated Ana o 1 were also subjected to in vitro pepsin digestion at pH 3.0 for 2 hr. Both glycosylated and deglycosylated Ana o 1 remained stable and reactive with IgE antibodies following digestion. PRACTICAL APPLICATION: Understanding the role of glycosylation in Ana o 1 immunoreactivity may provide insight into the potential development of hypoallergenic cashews/cashew products for sensitive individuals in the future.

Rapid liquid chromatography-mass spectrometry quantitation of glucose-regulating hormones from human islets of Langerhans.

Glucose homeostasis is maintained through the secretion of peptide hormones, such as insulin, somatostatin, and glucagon, from islets of Langerhans, clusters of endocrine cells found in the pancreas. This report describes an LC-MS method using multiple reaction monitoring for quantitation of insulin, C-peptide, glucagon, and somatostatin secretion from human islet populations. For rapid analysis, a 5 min separation was achieved using a 2.1 × 30 mm (i.d. x length) C18 column with 2.7 µm diameter core shell particles. A sacrificial protein hydrolysate was used with the sample and found to improve signal magnitude, repeatability, and to reduce carryover between runs. At optimized gradient conditions, the gradient run time was 4.55 min producing an average peak width of 0.3 min, a minimum resolution of 1.2, and a peak capacity of 20. As a proof of concept, the method was used to measure secretions from static incubations of human islets from 2 donors. Insulin and C-peptide were quantified and matched well with literature values of these hormones. We expect that this antibody-free quantitation of multiple hormones secreted from islets will provide insights into the temporal relationships of these peptides in the future.

D-serine mitigates cell loss associated with temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults, with an unknown etiology. A hallmark of TLE is the characteristic loss of layer 3 neurons in the medial entorhinal area (MEA) that underlies seizure development. One approach to intervention is preventing loss of these neurons through better understanding of underlying pathophysiological mechanisms. Here, we show that both neurons and glia together give rise to the pathology that is mitigated by the amino acid D-serine whose levels are potentially diminished under epileptic conditions. Focal administration of D-serine to the MEA attenuates neuronal loss in this region thereby preventing epileptogenesis in an animal model of TLE. Additionally, treatment with D-serine reduces astrocyte counts in the MEA, alters their reactive status, and attenuates proliferation and/or infiltration of microglia to the region thereby curtailing the deleterious consequences of neuroinflammation. Given the paucity of compounds that reduce hyperexcitability and neuron loss, have anti-inflammatory properties, and are well tolerated by the brain, D-serine, an endogenous amino acid, offers new hope as a therapeutic agent for refractory TLE.

Profiling Glucose-Stimulated and M3 Receptor-Activated Insulin Secretion Dynamics from Islets of Langerhans Using an Extended-Lifetime Fluorescence Dye.

Pulsatile insulin from pancreatic islets is crucial for glucose homeostasis, but the mechanism behind coordinated pulsatility is still under investigation. One hypothesis suggests that cholinergic stimulation of islets by pancreatic ganglia resets these endocrine units, producing synchronization. Previously, it was shown that intracellular Ca2+ oscillations within islets can be entrained by pulses of a cholinergic agonist, carbachol (CCh). Although these proxy measurements of Ca2+ provided insight into the synchronization mechanism, measurement of insulin output would be more direct evidence. To this end, a fluorescence anisotropy competitive immunoassay for online insulin detection from single and grouped islets in a microfluidic system was developed using a piezoelectric pressure-driven fluid delivery system and a squaraine rotaxane fluorophore, SeTau-647, as the fluorescent label for insulin. Due to SeTau-647 having a longer lifetime and higher brightness compared to the previously used Cy5 fluorophore, a 45% increase in the anisotropy range was observed with enhanced signal-to-noise ratio (S/N) of the measurements. This new system was tested by measuring glucose-stimulated insulin secretion from single and groups of murine and human islets. Distinct islet entrainment of groups of murine islets by pulses of CCh was also observed, providing further evidence for the hypothesis that pulsatile output from the ganglia can synchronize islet behavior. We expect that this relatively straightforward, homogeneous assay can be widely used for examining not only insulin secretion but other secreted factors from different tissues.

Synchronized stimulation and continuous insulin sensing in a microfluidic human Islet on a Chip designed for scalable manufacturing.

Pancreatic ß cell function is compromised in diabetes and is typically assessed by measuring insulin secretion during glucose stimulation. Traditionally, measurement of glucose-stimulated insulin secretion involves manual liquid handling, heterogeneous stimulus delivery, and enzyme-linked immunosorbent assays that require large numbers of islets and processing time. Though microfluidic devices have been developed to address some of these limitations, traditional methods for islet testing remain the most common due to the learning curve for adopting microfluidic devices and the incompatibility of most device materials with large-scale manufacturing. We designed and built a thermoplastic, microfluidic-based Islet on a Chip compatible with commercial fabrication methods, that automates islet loading, stimulation, and insulin sensing. Inspired by the perfusion of native islets by designated arterioles and capillaries, the chip delivers synchronized glucose pulses to islets positioned in parallel channels. By flowing suspensions of human cadaveric islets onto the chip, we confirmed automatic capture of islets. Fluorescent glucose tracking demonstrated that stimulus delivery was synchronized within a two-minute window independent of the presence or size of captured islets. Insulin secretion was continuously sensed by an automated, on-chip immunoassay and quantified by fluorescence anisotropy. By integrating scalable manufacturing materials, on-line, continuous insulin measurement, and precise spatiotemporal stimulation into an easy-to-use design, the Islet on a Chip should accelerate efforts to study and develop effective treatments for diabetes.

Chiral micellar electrokinetic chromatographic separation for determination of L- and D-primary amines released from murine islets of Langerhans.

D-amino acids have been located in various tissues including the endocrine portion of the pancreas, the islets of Langerhans. D-Serine (D-Ser), is of particular interest since it is an agonist for the ionotropic N-methyl-D-aspartate receptors. To examine the potential release of D-Ser and other D-amino acids from islets, a chiral micellar electrokinetic chromatography method was developed by derivatizing primary amines with 2,3-naphthalenedicarboxaldehyde and to achieve resolution of the enantiomers, two surfactants were used in the separation, sodium dodecyl sulfate and sodium deoxycholate. With the optimized conditions, 7 of 13 enantiomeric pairs that were tested had greater than baseline resolution, while the resolution of numerous other L-amino acids and small molecules were maintained. For the 17 compounds that were fully resolved, limits of detection were less than 10 nM. The resulting optimized separation method produced high efficiency peaks, with an average of 300,000 theoretical plates per peak and a peak capacity of 120. The method was used to examine the release of small molecules from groups of 50 murine islets of Langerhans. A peak was detected from islets incubated with 20 mM glucose that co-migrated with a D-Ser standard, although its level was below the quantifiable limit.

Gene expression patterns in synchronized islet populations.

In vivo levels of insulin are oscillatory with a period of ~5-10 minutes, indicating that the islets of Langerhans within the pancreas are synchronized. While the synchronizing factors are still under investigation, one result of this behavior is expected to be coordinated and oscillatory intracellular factors, such as intracellular Ca2+ levels, throughout the islet population. In other cell types, oscillatory intracellular signals, like intracellular Ca2+, have been shown to affect specific gene expression. To test how the gene expression landscape may differ between a synchronized islet population with its reproducible intracellular oscillations and an unsynchronized islet population with heterogeneous oscillations, gene set enrichment analysis (GSEA) was used to compare an islet population that had been synchronized using a glucose wave with a 5-min period, and an unsynchronized islet population. In the population exposed to the glucose wave, 58/62 islets showed synchronization as evidenced by coordinated intracellular Ca2+ oscillations with an average oscillation period of 5.1 min, while in the unsynchronized population 29/62 islets showed slow oscillations with an average period of 5.2 min. The synchronized islets also had a significantly smaller drift of their oscillation period during the experiment as compared to the unsynchronized population. GSEA indicated that the synchronized population had reduced expression of gene sets related to protein translation, protein turnover, energy expenditure, and insulin synthesis, while those that were related to maintenance of cell morphology were increased.

Online Measurement of Glucose Consumption from HepG2 Cells Using an Integrated Bioreactor and Enzymatic Assay.

Hepatocytes help to maintain glucose homeostasis in response to a variety of signals, including pancreatic hormones such as insulin. Insulin is released from the pancreas with variable dynamics, yet the role that these play in regulating glucose metabolism in the liver is still unclear. In this study, a modular microfluidic system was developed to quantitatively measure the effect of insulin dynamics on glucose consumption by a human hepatocarcinoma cell line, HepG2. A microfluidic bioreactor that contained 106 HepG2 cells was cultured for up to 10 days in an incubator. For glucose consumption experiments, the bioreactor was removed from the incubator and connected with reagents for an enzymatic glucose assay. The mixed components were then delivered into a droplet-based microfluidic system where the intensity of the fluorescent product of the enzyme assay was used to quantify the glucose concentration. By optimizing the mixing time of the reagents, the dynamic range of the enzymatic assay was adjusted to 0-12 mM glucose and had a time resolution of 96 ± 12 s. The system was used to observe rapid changes in insulin-induced glucose consumption from HepG2 cells. This assay format is versatile and can be expanded to measure a variety of hepatic metabolites, such as lactate, pyruvate, or ketone bodies, which will enable the correlation of pancreatic hormone dynamics to liver metabolism.