Discovery of KIN-3248, An Irreversible, Next Generation FGFR Inhibitor for the Treatment of Advanced Tumors Harboring FGFR2 and/or FGFR3 Gene Alterations.

Fibroblast growth factor receptor (FGFR) alterations are present as oncogenic drivers and bypass mechanisms in many forms of cancer. These alterations can include fusions, amplifications, rearrangements, and mutations. Acquired drug resistance to current FGFR inhibitors often results in disease progression and unfavorable outcomes for patients. Genomic profiling of tumors refractory to current FGFR inhibitors in the clinic has revealed several acquired driver alterations that could be the target of next generation therapeutics. Herein, we describe how structure-based drug design (SBDD) was used to enable the discovery of the potent and kinome selective pan-FGFR inhibitor KIN-3248, which is active against many acquired resistance mutations. KIN-3248 is currently in phase I clinical development for the treatment of advanced tumors harboring FGFR2 and/or FGFR3 gene alterations.

The Discovery of Exarafenib (KIN-2787): Overcoming the Challenges of Pan-RAF Kinase Inhibition.

RAF, a core signaling component of the MAPK kinase cascade, is often mutated in various cancers, including melanoma, lung, and colorectal cancers. The approved inhibitors were focused on targeting the BRAFV600E mutation that results in constitutive activation of kinase signaling through the monomeric protein (Class I). However, these inhibitors also paradoxically activate kinase signaling of RAF dimers, resulting in increased MAPK signaling in normal tissues. Recently, significant attention has turned to targeting RAF alterations that activate dimeric signaling (class II and III BRAF and NRAS). However, the discovery of a potent and selective inhibitor with biopharmaceutical properties suitable to sustain robust target inhibition in the clinical setting has proven challenging. Herein, we report the discovery of exarafenib (15), a highly potent and selective inhibitor that intercepts the RAF protein in the dimer compatible αC-helix-IN conformation and demonstrates anti-tumor efficacy in preclinical models with BRAF class I, II, and III and NRAS alterations.

Design and validation of an exposure system for efficient inter-animal SARS-CoV-2 airborne transmission in Syrian hamsters.

IMPORTANCE: The main route of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission is airborne. However, there are few experimental systems that can assess the airborne transmission dynamics of SARS-CoV-2 in vivo. Here, we designed, built, and characterized a hamster transmission caging and exposure system that allows for efficient SARS-CoV-2 airborne transmission in Syrian hamsters without contributions from fomite or direct contact transmission. We successfully measured SARS-CoV-2 viral RNA in aerosols and demonstrated that SARS-CoV-2 is transmitted efficiently at either a 1:1 or 1:4 infected index to naïve recipient hamster ratio. This is meaningful as a 1:4 infected index to naïve hamster ratio would allow for simultaneous comparisons of various interventions in naïve animals to determine their susceptibility to infection by aerosol transmission of SARS-CoV-2. Our SARS-CoV-2 exposure system allows for testing viral airborne transmission dynamics and transmission-blocking therapeutic strategies against SARS-CoV-2 in Syrian hamsters.

Leveraging a smartphone to perform time-gated luminescence measurements.

Empowered by advanced on-board sensors, high-performance optics packages and ever-increasing computational power, smartphones have democratized data generation, collection, and analysis. Building on this capacity, many platforms have been developed to enable its use as an optical sensing platform for colorimetric and fluorescence measurements. In this paper, we report the ability to enable a smartphone to perform laboratory quality time-resolved analysis of luminescent samples via the exploitation of the rolling shutter mechanism of the native CMOS imager. We achieve this by leveraging the smartphone's standard image capture applications, commercially available image analysis software, and housing the device within a UV-LED containing case. These low-cost modifications enable us to demonstrate the smartphone's analytical potential by performing tasks ranging from authentication and encryption to the interrogation of packaging, compounds, and physical phenomena. This approach underscores the power of repurposing existing technologies to extend the reach and inclusivity of scientific exploration, opening new avenues for data collection and analysis.

Inhalation delivery of nucleic acid gene therapies in preclinical drug development.

INTRODUCTION: Inhaled gene therapy programs targeting diseases of the lung have seen increasing interest in recent years, though as of yet no product has successfully entered the market. Preclinical research to support such programs is critically important in maximizing the chances of developing successful candidates. AREAS COVERED: Aspects of inhalation delivery of gene therapies are reviewed, with a focus on preclinical research in animal models. Various barriers to inhalation delivery of gene therapies are discussed, including aerosolization stresses, aerosol behavior in the respiratory tract, and disposition processes post-deposition. Important aspects of animal models are considered, including determinations of biologically relevant determinations of dose and issues related to translatability. EXPERT OPINION: Development of clinically-efficacious inhaled gene therapies has proven difficult owing to numerous challenges. Fit-for-purpose experimental and analytical methods are necessary for determinations of biologically relevant doses in preclinical animal models. Further developments in disease-specific animal models may aid in improving the translatability of results in future work, and we expect to see accelerated interests in inhalation gene therapies for various diseases. Sponsors, researchers, and regulators are encouraged to engage in early and frequent discussion regarding candidate therapies, and additional dissemination of preclinical methodologies would be of immense value in avoiding common pitfalls.

Observed Reductions in the Infectivity of Bioaerosols Containing Bovine Coronavirus Under Repetitively Pulsed RF Exposure.

OBJECTIVE: The purpose of the present study is to investigate the inactivation of bioaerosols containing Bovine Coronavirus, BCoV, under repetitively pulsed radio frequency (RF) electromagnetic exposure. METHODS: These experiments were performed in a waveguide containing a flowing aerosol stream and were limited to a single RF waveform: ∼2 µs square envelope, 5.6 GHz, 4.8 kHz repetition rate. Aerosol streams were exposed to RF electric field amplitudes in the range of 41.9 +/-6.2 kV/m. Under laminar flow conditions, 75% of the total collected aerosol stream spends 0.85 seconds or less in the RF exposure region. RESULTS: Application of the RF waveform changed mean survival rate of the aerosolized BCoV by -0.58 decades (roughly a 74% reduction) and impacted the variance and standard deviation of the experimental results, with the RF exposure data showing an 800% increase in variance and 196% increase in standard deviation over the control results. Experimental results were compared to those from an analytic electromagnetic-heating inactivation model. CONCLUSION: The comparison indicated the feasibility that the observed reduction in BCoV survival rate might be due to a combination of thermal effects and non-thermal electric field effects. SIGNIFICANCE: Developing better insight into the mechanisms of inactivation is important for understanding the potential limits of efficacy for this method. Additionally, these results contribute an important baseline for the impact of electromagnetic fields on aerosolized pathogens.

Spontaneous Coronary Artery Dissection and Its Management: A Case Report.

We illustrate a notable case of an elderly male presenting to a community hospital with six out of 10 substernal chest discomfort and electrocardiogram changes consistent with an anterolateral myocardial infarction. Percutaneous Coronary Intervention (PCI) was initiated following aspirin and anticoagulation administration, which further revealed a critical distal left main spontaneous coronary artery dissection (SCAD). Cardiothoracic and Vascular Surgery consults led to the recommendation of emergent two-vessel Coronary Artery Bypass Grafting (CABG). The patient's clinical status resolved to full recovery and was discharged on postoperative day five. The incidence of SCAD in older men has not been well documented in current literature. Prevalence in older males is 0.02%. However, it rises to 10.8% in females less than 50 years of age and with acute coronary syndromes (ACS) and ST-segment elevation. Our aim is to incorporate this case report into the current literature and help improve early diagnosis and treatment based on current recommendations.

Exploring antiviral and anti-inflammatory effects of thiol drugs in COVID-19.

The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for the binding of SARS-2-S to angiotensin-converting enzyme 2 (ACE2), suggesting that drugs with a functional thiol group ("thiol drugs") may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore the antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection, but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury, and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.

Halo-A Universal Fluorescence Reader Based Threat Agent Detection Platform-A Proof of Concept Study Using SARS-CoV-2 Assays.

Polymerase chain reaction (PCR) remains the gold standard in disease diagnostics due to its extreme sensitivity and specificity. However, PCR tests are expensive and complex, require skilled personnel and specialized equipment to conduct the tests, and have long turnaround times. On the other hand, lateral flow immunoassay-based antigen tests are rapid, relatively inexpensive, and can be performed by untrained personnel at the point of care or even in the home. However, rapid antigen tests are less sensitive than PCR since they lack the inherent target amplification of PCR. It has been argued that rapid antigen tests are better indicators of infection in public health decision-making processes to test, trace, and isolate infected people to curtail further transmission. Hence, there is a critical need to increase the sensitivity of rapid antigen tests and create innovative solutions to achieve that goal. Herein, we report the development of a low-cost diagnostic platform, enabling rapid detection of SARS-CoV-2 under field or at-home conditions. This platform (Halo™) is a small, highly accurate, consumer-friendly diagnostic reader paired with fluorescently labeled lateral flow assays and custom software for collection and reporting of results. The focus of this study is to compare the analytical performance of HaloTM against comparable tests that use either colloidal gold nanoparticles or fluorescence-based reporters in simulated nasal matrix and not in clinical samples. Live virus data has demonstrated limit of detection performance of 1.9 TCID50/test in simulated nasal matrix for the delta variant, suggesting that single-assay detection of asymptomatic SARS-CoV-2 infections may be feasible. Performance of the system against all tested SARS CoV-2 virus variants showed comparable sensitivities indicating mutations in SARS-CoV-2 variants do not negatively impact the assay.

Effects of soluble guanylate cyclase stimulator on renal function in ZSF-1 model of diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is associated with endothelial dysfunction and oxidative stress, in which the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway is impaired. We hypothesize that sGC stimulator Compound 1 can enhance NO signaling, reduce proteinuria in a diabetic nephropathy preclinical model with diminished NO bioavailability and increased oxidized sGC. Therefore, we evaluated the effect of sGC stimulator Compound 1 on the renal effect in obese ZSF1 (ZSF1 OB) rats. MATERIALS AND METHODS: The sGC stimulator Compound 1, the standard of care agent Enalapril, and a combination of Compound 1 and Enalapril were administered chronically to obese ZSF1 rats for 6 months. Mean arterial pressure, heart rate, creatinine clearance for glomerular filtration rate (eGFR), urinary protein excretion to creatinine ratio (UPCR), and urinary albumin excretion ratio (UACR) were determined during the study. The histopathology of glomerular and interstitial lesions was assessed at the completion of the study. RESULTS: While both Compound 1 and Enalapril significantly reduced blood pressure, the combination of Compound 1 and Enalapril normalized blood pressure levels. Compound 1 improved eGFR and reduced UPCR and UACR. A combination of Enalapril and Compound 1 resulted in a marked reduction in UPCR and UACR and improved GFR. CONCLUSION: The sGC stimulator Compound 1 as a monotherapy slowed renal disease progression, and a combination of the sGC stimulator with Enalapril provided greater renal protection in a rodent model of diabetic nephropathy.

Nonlinear transmission line-driven apparatus for short-pulse microwave exposure of aerosolized pathogens.

A system capable of exposing a flowing aerosol stream to short duration (2-4 ns), high-power RF waveforms is described. The system utilizes a C-band gyromagnetic nonlinear transmission line source having peak power outputs ranging as high as 80 kW at a center frequency of 4.2 GHz. RF electric field magnitudes of up to 280 kV/m ± 17% are achieved within the aerosol flow region of the RF exposure apparatus.

Apparatus for controlled microwave exposure of aerosolized pathogens.

A set of three apparatus enabling RF exposure of aerosolized pathogens at four chosen frequencies (2.8 GHz, 4.0 GHz, 5.6 GHz, and 7.5 GHz) has been designed, simulated, fabricated, and tested. Each apparatus was intended to operate at high power without leakage of RF into the local environment and to be compact enough to fit within biocontainment enclosures required for elevated biosafety levels. Predictions for the range of RF electric field exposure, represented by the complex electric field vector magnitude, that an aerosol stream would be expected to encounter while passing through the apparatus are calculated for each of the chosen operating frequencies.

Thiol drugs decrease SARS-CoV-2 lung injury in vivo and disrupt SARS-CoV-2 spike complex binding to ACE2 in vitro.

Neutrophil-induced oxidative stress is a mechanism of lung injury in COVID-19, and drugs with a functional thiol group ("thiol drugs"), especially cysteamine, have anti-oxidant and anti-inflammatory properties that could limit this injury. Thiol drugs may also alter the redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) and thereby disrupt ACE2 binding. Using ACE2 binding assay, reporter virus pseudotyped with SARS-CoV-2 spikes (ancestral and variants) and authentic SARS-CoV-2 (Wuhan-1), we find that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus entry into cells. Pseudoviruses carrying variant spikes were less efficiently inhibited as compared to pseudotypes bearing an ancestral spike, but the most potent drugs still inhibited the Delta variant in the low millimolar range. IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. In hamsters infected with SARS-CoV-2, intraperitoneal (IP) cysteamine decreased neutrophilic inflammation and alveolar hemorrhage in the lungs but did not decrease viral infection, most likely because IP delivery could not achieve millimolar concentrations in the airways. These data show that thiol drugs inhibit SARS-CoV-2 infection in vitro and reduce SARS-CoV-2-related lung injury in vivo and provide strong rationale for trials of systemically delivered thiol drugs as COVID-19 treatments. We propose that antiviral effects of thiol drugs in vivo will require delivery directly to the airways to ensure millimolar drug concentrations and that thiol drugs with lower thiol pKa values are most likely to be effective.

100 m Long Thermally Drawn Supercapacitor Fibers with Applications to 3D Printing and Textiles.

Supercapacitor fibers, with short charging times, long cycle lifespans, and high power densities, hold promise for powering flexible fabric-based electronics. To date, however, only short lengths of functioning fiber supercapacitors have been produced. The primary goal of this study is to introduce a supercapacitor fiber that addresses the remaining challenges of scalability, flexibility, cladding impermeability, and performance at length. This is achieved through a top-down fabrication method in which a macroscale preform is thermally drawn into a fully functional energy-storage fiber. The preform consists of five components: thermally reversible porous electrode and electrolyte gels; conductive polymer and copper microwire current collectors; and an encapsulating hermetic cladding. This process produces 100 m of continuous functional supercapacitor fiber, orders of magnitude longer than any previously reported. In addition to flexibility (5 mm radius of curvature), moisture resistance (100 washing cycles), and strength (68 MPa), these fibers have an energy density of 306 µWh cm-2 at 3.0 V and ≈100% capacitance retention over 13 000 cycles at 1.6 V. To demonstrate the utility of this fiber, it is machine-woven and used as filament for 3D printing.

Build Insight, Change Thinking, Inform Action: Considerations for Increasing the Number of Indigenous Students in Canadian Physical Therapy Programmes.

Purpose: We explored the perspectives of experts on increasing the recruitment of Indigenous students into Canadian physical therapy (PT) programmes. Methods: For this qualitative interpretivist study, we conducted in-depth, semi-structured interviews with individuals with expertise in encouraging Indigenous students to pursue higher education, recruiting them into PT programmes, or both. Data were organized using NVivo and analyzed using the DEPICT method, which included inductive and deductive coding to develop broader themes. Results: Analyzing the participants' perspectives revealed three themes, which could be layered sequentially, so that each informed the next: (1) building insight by increasing awareness of structural forces and barriers; (2) changing thinking, using a paradigm shift, from the dominant Eurocentric orientation to a view that respects the sovereignty and self-determination of Indigenous peoples; and (3) informing action by recommending practical strategies to facilitate the recruitment of Indigenous students into Canadian PT programmes. Conclusions: This is the first study to provide evidence of the structural considerations, barriers to, and facilitators of increasing the recruitment of Indigenous students into Canadian PT programmes.

Objectif : explorer les points de vue des experts pour recruter plus d'étudiants autochtones au sein des programmes de physiothérapie canadiens. Méthodologie : dans le cadre de cette étude d'interprétation qualitative, les chercheurs ont réalisé des entrevues semi-structurées approfondies avec des personnes qui possèdent des compétences pour encourager les étudiants autochtones à faire des études supérieures et pour les recruter dans des programmes de physiothérapie. Ils ont organisé les données à l'aide du logiciel NVivo et les ont analysées à l'aide de la méthode DEPICT, qui inclut un codage inductif et déductif pour établir des thèmes plus vastes. Résultats : l'analyse des points de vue des participants a fait ressortir trois thèmes, qui pourraient être superposés séquentiellement pour que chacun éclaire le suivant, comme suit : 1) favoriser les prises de conscience en faisant mieux connaître les forces et obstacles structurels; 2) faire évoluer les mentalités par un changement de paradigme afin de passer de l'orientation eurocentrique dominante à une vision qui respecte la souveraineté et l'autodétermination des peuples autochtones et 3) éclairer les actions en recommandant des stratégies pratiques pour faciliter le recrutement d'étudiants autochtones au sein des programmes de physiothérapie canadiens. Conclusions : c'est la première étude à fournir des données probantes sur les facteurs structurels, les obstacles et les incitations liés au recrutement d'un plus grand nombre d'étudiants autochtones au sein des programmes de physiothérapie canadiens.

Diode fibres for fabric-based optical communications.

Semiconductor diodes are basic building blocks of modern computation, communications and sensing1. As such, incorporating them into textile-grade fibres can increase fabric capabilities and functions2,  to encompass, for example,  fabric-based communications or physiological monitoring. However, processing challenges have so far precluded the realization of semiconducting diodes of high quality in thermally drawn fibres. Here we demonstrate a scalable thermal drawing process of electrically connected diode fibres. We begin by constructing a macroscopic preform that hosts discrete diodes internal to the structure alongside hollow channels through which conducting copper or tungsten wires are fed. As the preform is heated and drawn into a fibre, the conducting wires approach the diodes until they make electrical contact, resulting in hundreds of diodes connected in parallel inside a single fibre. Two types of in-fibre device are realized: light-emitting and photodetecting p-i-n diodes. An inter-device spacing smaller than 20 centimetres is achieved, as well as light collimation and focusing by a lens designed in the fibre cladding. Diode fibres maintain performance throughout ten machine-wash cycles, indicating the relevance of this approach to apparel applications. To demonstrate the utility of this approach, a three-megahertz bi-directional optical communication link is established between two fabrics containing receiver-emitter fibres. Finally, heart-rate measurements with the diodes indicate their potential for implementation in all-fabric physiological-status monitoring systems. Our approach provides a path to realizing ever more sophisticated functions in fibres, presenting  the prospect of a fibre 'Moore's law' analogue  through the increase of device density and function in thermally drawn textile-ready fibres.

An integrin antagonist (MK-0429) decreases proteinuria and renal fibrosis in the ZSF1 rat diabetic nephropathy model.

Multiple integrins have been implicated in modulating renal function. Modulation of integrin function can lead to pathophysiological processes associated with diabetic nephropathy such as alterations in the glomerular filtration barrier and kidney fibrosis. The complexity of these pathophysiological changes implies that multiple integrin subtypes might need to be targeted to ameliorate the progression of renal disease. To address this hypothesis, we investigated the effects of MK-0429, a compound that was originally developed as an αvß3 inhibitor for the treatment of osteoporosis, on renal function and fibrosis. We demonstrated that MK-0429 is an equipotent pan-inhibitor of multiple av integrins. MK-0429 dose-dependently inhibited podocyte motility and also suppressed TGF-ß-induced fibrosis marker gene expression in kidney fibroblasts. Moreover, in the obese ZSF1 rat model of diabetic nephropathy, chronic treatment with MK-0429 resulted in significant reduction in proteinuria, kidney fibrosis, and collagen accumulation. In summary, our results suggest that inhibition of multiple integrin subtypes might lead to meaningful impact on proteinuria and renal fibrosis in diabetic nephropathy.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

Pyrolyzable Nanoparticle Tracers for Environmental Interrogation and Monitoring.

Environmental tracing applications require materials that can be detected in complex fluids composed of multiple phases and contaminants. Moreover, large libraries of tracers are necessary in order to mitigate memory effects and to deploy multiple tracers simultaneously in complex oil fields. Herein, we disclose a novel approach based on the thermal decomposition of polymeric nanoparticles comprised of styrenic and methacrylic monomers. Polymeric nanoparticles derived from these monomers cleanly decompose into their constituent monomers at elevated temperatures, thereby maximizing atom economy wherein the entire nanoparticle mass contributes to the generation of detectable units. A total of ten unique single monomer particles and three dual-monomer particles were synthesized using semicontinuous monomer starved addition polymerization. The pyrolysis gas chromatography-flame ionization detection/mass spectrometry (GC-FID/MS) behavior of these particles was studied using high-pressure mass spectrometry. The programmable nature of our methodology permits simultaneous removal of contaminants and subsequent identification and quantification in a single analytical step.

Design, Synthesis, and Evaluation of Novel and Selective G-protein Coupled Receptor 120 (GPR120) Spirocyclic Agonists.

Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effective insulin sensitizers, absent of weight gain, has been a long-standing goal of diabetes research. G-protein coupled receptor 120 (GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic agonists have been associated with insulin sensitizing, anti-inflammatory, and fat metabolism effects. Herein we describe the design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance (HOMA-IR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp in diet-induced obese (DIO) mice.