Copper is a trace element whose electronic configuration provides it with essential structural and catalytic functions. However, in excess, both its high protein affinity and redox-catalyzing properties can lead to hazardous consequences. In addition to promoting oxidative stress, copper is gaining interest for its effects on neurotransmission through modulation of GABAergic and glutamatergic receptors and interaction with the dopamine reuptake transporter. The aim of the present study was to investigate the effects of copper overexposure on the levels of dopamine, noradrenaline, and serotonin, or their main metabolites in rat\'s striatum extracellular fluid. Copper was injected intraperitoneally using our previously developed model, which ensured striatal overconcentration (2 mg CuCl2/kg for 30 days). Subsequently, extracellular fluid was collected by microdialysis on days 0, 15, and 30. Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and noradrenaline (NA) levels were then determined by HPLC coupled with electrochemical detection. We observed a significant increase in the basal levels of DA and HVA after 15 days of treatment (310% and 351%), which was maintained after 30 days (358% and 402%), with no significant changes in the concentrations of 5-HIAA, DOPAC, and NA. Copper overload led to a marked increase in synaptic DA concentration, which could contribute to the psychoneurological alterations and the increased oxidative toxicity observed in Wilson\'s disease and other copper dysregulation states.

Cell secretion repairs tissue damage and restores homeostasis throughout adult life. The extracellular heat shock protein-90alpha (eHsp90α) has been reported as an exosome cargo and a potential driver of wound healing. However, neither the mechanism of secretion nor the genetic evidence for eHsp90α in wound healing has been substantiated. Herein, we show that tissue injury causes massive deposition of eHsp90α in tissues and secretion of eHsp90α by cells. Sequential centrifugations of conditioned medium from relevant cell lines revealed the relative distributions of eHsp90α in microvesicle, exosome and trypsin-sensitive supernatant fractions to be approximately <2%, <4% and >95%, respectively. Establishing the cell-number-to-interstitial-fluid-volume (CIF) ratio for the microenvironment of human tissues as 1 × 109 cells: 1 mL interstitial fluid enabled us to predict the corresponding tissue concentrations of eHsp90α in these fractions as 3.74 μg/mL, 5.61 μg/mL and 178 μg/mL. Remarkably, the 178 μg/mL eHsp90α matches the previously reported 100-300 μg/mL of recombinant eHsp90α whose topical application promotes maximum wound healing in animal models. More importantly, we demonstrate that two parallel secretory autophagy-regulating gene families, the autophagy-regulating (AR) genes and the Golgi reassembly-stacking protein (GRASP) genes work together to mediate the secretion of the physiological concentration of eHsp90α to promote wound healing. Thus, utilization of the CIF ratio-based extrapolation method may enable investigators to rapidly predict biomarker targets from cell-conditioned-medium data.

Diseases mediated by cytokine storms are often characterized by an overexuberant pace of pathogenesis accompanied by significant morbidity and mortality. Thus, near real-time (NRT) detections via a site-of-inflammation (SOI) sampling of proinflammatory cytokines are essential to ensure a timely and effective treatment of acute inflammations, which up to now, has not been fully possible. In this work, we proposed a novel NRT and SOI immunosensor using ZIF-8 signal amplification together with an off-on strategy. To achieve NRT detections via a SOI sampling, the body fluid of choice is the dermal interstitial fluid (ISF). The significant merits of ISF over blood are the quality, quantity and diversity of ISF-based biomarkers; the fluid is non-coagulating, making it feasible to perform multiple or continuous samplings and the sampling is minimally invasive. Our immunosensor requires only 5 μL of ISF to achieve a simultaneous detection of five highly potent proinflammatory cytokines: IL-6, IFN-γ, IL-1β, TNF-α, IP-10. We employed a microneedle array patch (MAP) together with a trifurcated nozzle pump to extract a mean volume of between 30 and 60 μL of ISF in 20 min. Under optimal conditions, the biosensor is capable of high-quality performance that exhibits a lower limit of detection (LOD) of 5.761 pg/mL over a wide linear range of 5.761-3 ‒ 20.00 ng/mL. We believe our immunosensor for NRT detections via a SOI sampling of ISF-biomarkers offers new theranostic opportunities that may not be possible with blood-based biomarkers.

Diabetes is a chronic disease with significant complications, necessitating regular treatment and checkups, which can be costly and time-consuming for patients. To address this, we developed the Sliding Microneedle (MN)-Lateral flow immunoassay strip (LFIAs) device that combines the advantages of MNs and LFIAs to detect IL-6, an independent biomarker for diabetes complications. This device offers rapid and highly sensitive detection of IL-6 by extracting interstitial fluid (ISF) through MNs and transferring it to LFIAs. The stainless MN, embedded in the 3D-printed Sliding MN-LFIAs device, was inserted into the skin at a 20° angle, minimizing blood contamination risk. With a filter paper attached to the MN surface, the device collected 4.65 ± 0.05 μL of ISF containing IL-6 within 90 s. The ISF was then transferred to the LFIAs using a running buffer. After a 15-min reaction, silver enhancement (SE) treatment was applied, allowing for the highly sensitive and specific detection of IL-6 at 102 pg/mL concentrations. The Sliding MN-LFIAs device successfully distinguished between normal and diabetic rat models, demonstrating its potential as an effective tool for detecting diabetes complications quickly and affordably.

UNASSIGNED: Lymphatic valves are specialized structures in collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves. Conventionally, in many cell types, AKT is phosphorylated at Ser473 by the mTORC2 (mammalian target of rapamycin complex 2). However, mTORC2 has not yet been implicated in lymphatic valve formation.
UNASSIGNED: In vivo and in vitro techniques were used to investigate the role of Rictor, a critical component of mTORC2, in lymphatic endothelium.
UNASSIGNED: Here, we showed that embryonic and postnatal lymphatic deletion of Rictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human dermal lymphatic endothelial cells not only reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions but also abolished the upregulation of AKT activity and valve-forming genes in response to oscillatory shear stress. We further showed that the AKT target, FOXO1 (forkhead box protein O1), a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric lymphatic endothelial cells in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in lymphatic vessels of the ear and mesentery.
UNASSIGNED: Our work identifies a novel role for RICTOR in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately enables the formation and maintenance of lymphatic valves.

Congestive heart failure (CHF) is a fatal disease with progressive severity and no cure; the heart\'s inability to adequately pump blood leads to fluid accumulation and frequent hospital readmissions after initial treatments. Therefore, it is imperative to continuously monitor CHF patients during its early stages to slow its progression and enable timely medical interventions for optimal treatment. An increase in interstitial fluid pressure (IFP) is indicative of acute CHF exacerbation, making IFP a viable biomarker for predicting upcoming CHF if continuously monitored. In this paper, we present an inductor-capacitor (LC) sensor for subcutaneous wireless and continuous IFP monitoring. The sensor is composed of inexpensive planar copper coils defined by a simple craft cutter, which serves as both the inductor and capacitor. Because of its sensing mechanism, the sensor does not require batteries and can wirelessly transmit pressure information. The sensor has a low-profile form factor for subcutaneous implantation and can communicate with a readout device through 4 layers of skin (12.7 mm thick in total). With a soft silicone rubber as the dielectric material between the copper coils, the sensor demonstrates an average sensitivity as high as -8.03 MHz/mmHg during in vitro simulations.

Diabetes is a common chronic metabolic disease with a wide range of clinical symptoms and consequences and one of the main causes of death. For the management of diabetes, painless and continuous interstitial fluid (ISF) glucose monitoring is ideal. Here, we demonstrate continuous diabetes monitoring using an integrated microneedle (MN) biosensor with an emergency alert system. MNs are a novel technique in the field of biomedical engineering because of their ability to analyze bioinformation with minimal invasion. In this work we developed a poly(methyl methacrylate) (PMMA) based MN glucose sensor. The device was produced by the 3D printing technique, microfabrication, electrodeposition, and enzyme immobilization step. The in vitro test for the glucose MN sensor showed a linear range from 1.5 to 14 mM with a sensitivity of 1.51 μA mM-1, limit of detection (LOD) of 0.35 mM and good selectivity. Highly repeatable sensing is observed with good reproducibility. The interference-free detection of glucose in the presence of physiologically relevant concentrations of ascorbic acid, uric acid, and mannose is demonstrated, along with the operational stability of the array. After resolving the biofouling consequences linked to on-body sensing, this MN platform would be appealing for minimally invasive electrochemical glucose monitoring. An alert is sent to confidants via email or SMS when the values are abnormal. The application is also able to display the recorded values in the form of a graph to help determine the state of health of the user over a period of time. It can be concluded that continuous monitoring and an emergency alert system are important for keeping an eye on diabetic patients and can send alert in case of an abnormal situation of the patient.

This study explored the impact of varying energy availability (EA) on the 24-h interstitial fluid glucose concentration (IGC) in five elite male Japanese triathletes at a training camp. Measurements of IGC, energy and macronutrient intake, and exercise energy expenditure (EEE) through metabolic equivalents (METs) from training logs were conducted. Three subjects were evaluated over two 4-day periods, and two subjects over one 4-day period. Findings revealed significant correlations of daily mean nocturnal IGC with daily EA (r = 0.553, p = 0.001) and energy intake (EI) (r = 0.595, p < 0.001). However, no significant correlation was found between mean daily nocturnal IGC and EEE (r = -0.278, p = 0.124). Daytime IGC was ≥110 mg/dL for >50% of the time in all subjects, except on 1 day in one subject, and never fell <70 mg/dL. Therefore, daily EA may influence nocturnal IGC in elite male triathletes, although high daytime IGC levels were maintained without hypoglycemia.

Pathological proteins including tau are produced in neurons and released into interstitial fluid (ISF) in a neural activity-dependent manner during wakefulness. Pathological proteins in ISF can be removed from the brain via the glymphatic pathway during nighttime. Thus, in individuals with Alzheimer\'s disease (AD) that have dysregulated sleep/wake rhythm, application of orexin receptor 2 (OX2R) agonists during daytime could recover the efflux of pathological proteins to ISF and indirectly promote the glymphatic pathway by improving the quality of nighttime sleep after proper daytime arousal, resulting in increased removal of these proteins from the brain. We investigated this hypothesis using OX-201, a novel OX2R-selective agonist with a 50% effective concentration of 8.0 nM. Diurnal rhythm of tau release into hippocampal ISF correlated well with neuronal activity and wakefulness in wild-type mice. In both wild-type and human P301S tau transgenic mice, OX-201 induced wakefulness and promoted tau release into hippocampal ISF. Human P301S tau transgenic mice, tested under our conditions, showed longer wakefulness time, which differs from individuals with AD. OX-201 treatment over 2 months did not alter hippocampal tau levels. Although further studies are required, at a minimum OX2R agonists may not exacerbate tau accumulation in individuals with tauopathy, including AD.

Dupilumab is a monoclonal antibody approved for the treatment of atopic dermatitis (AD); however, its effects on molecular, cellular, and immunological levels remain to be elucidated. In this study, blood and dermal interstitial fluid (ISF) from nonlesional (NL) and lesional (L) skin were collected from eight patients with moderate to severe AD, before (visit 2-v2) and at the end of a 16-week treatment with dupilumab (visit 10-v10). Clinical treatment effect was demonstrated by significantly decreased AD severity scores at the end of treatment. At v10 versus v2, the percentages of CD4+ interleukin-producing cells showed a decreasing trend in ISF L and NL, unbound IL-4 levels in plasma were increased, IL-5 levels in ISF L reduced, and levels of factors involved in anti-inflammatory pathways and re-epithelization increased. At v2, ISF L showed that AD lesions might have altered amino acid pathways and lipid signaling compared to ISF NL. At v10, ISF L exhibited raised levels of long- and very-long-chain fatty acids and lipids compared to v2. Furthermore, dupilumab administration caused reduced expression of miR-155-5p and miR-378a-3p in ISF L. In conclusion, results from the present study provided novel knowledge by linking local immune and metabolic alterations to AD pathogenesis and treatment response.