Bifunctional small molecules that mediate the degradation of extracellular proteins.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

ACEstat: A DIY Guide to Unlocking the Potential of Integrated Circuit Potentiostats for Open-Source Electrochemical Analysis.

Miniaturization of analytical instrumentation is paramount to enabling convenient in-field sensing. The recent thrust in potentiostat miniaturization for electrochemical sensing and general use has led to the development of commercial application specific integrated circuits (ASICs) that pack all the power of a benchtop instrument into one 5 mm × 5 mm chip. While the capabilities of these integrated circuits far exceed those of open-source potentiostats in the literature, the activation barrier for their implementation requires extensive electrical and software engineering expertise to overcome. In order to more rapidly bring the utility of ASIC potentiostats to researchers, we present a low size, weight, power, and cost (Low SWaP-C) Army Corps of Engineers potentiostat (ACEstat) based on the widely available ADuCM355 offered by Analog Devices. This potentiostat is a streamlined and fully programmable device that leverages industry-leading integrated hardware to perform electrochemical measurements such as cyclic voltammetry, pulse voltammetry, and electrochemical impedance spectroscopy. The ACEstat enables control over a wide range of test parameters and displays results through an intuitive, open-source graphical user interface available on mobile devices and computers. In this report, we present an approachable, do-it-yourself guide to unlocking the capabilities of this integrated circuit potentiostat by outlining the fabrication and programming details necessary to facilitate electroanalysis. Furthermore, we demonstrate the practicality of this device by detecting 2,4,6-trinitrotoluene (TNT) in water at sub-mg/L detection limits, highlighting its potential for in-field use.

Selecting an Optimal Faraday Cage To Minimize Noise in Electrochemical Experiments.

The ubiquitous Faraday cage, an experimental component particularly essential for nanoelectrochemical measurements, is responsible for neutralizing noise introduced by electromagnetic interference (EMI). Faraday cage designs abound in the literature, often exhibiting varying thicknesses, mesh sizes, and base materials. The fact that the Faraday cage composition most often goes unreported underscores the fact that many electrochemical researchers assume a 100% EMI reduction for any given design. In this work, this assumption is challenged from a theoretical and empirical perspective by highlighting the physical principles producing the Faraday effect. A brief history of the Faraday cage and a simplified theoretical approach introduce fundamental considerations regarding optimal design properties. In practice, time-domain noise profiles and corresponding Fourier transform frequency domain information for custom-built Faraday cages reveal that maximally conductive cages provide more optimal EMI exclusion.

A Generalized Potentiostat Adaptor for Multiplexed Electroanalysis.

Electrochemical measurements over an array of electrodes may be accomplished with one of three potentiostat architectures: a single-channel device which averages the signal from a number of interconnected electrodes, a multichannel device with dedicated circuits for each electrode, or a single-channel device with a multiplexer interface to isolate the signal from specific electrodes. Of these three architectures, the use of a multiplexer interface is best suited to facilitate measurements over individual electrodes without the need for large numbers of dedicated potentiostat channels. We present a versatile strategy for the development of flexible printed circuit (FPC) electrode arrays with accompanying multiplexing hardware to interface with single-channel potentiostats. The FPC array was fabricated with 78 individually addressable 0.3 mm diameter gold working electrodes and characterized using optical and scanning electron microscopy, energy dispersive spectroscopy, profilometry, impedance spectroscopy, and cyclic voltammetry to investigate the morphology, elemental composition, height profile, impedance characteristics, and electrochemical response, respectively. Interfacing the FPC array via a simple connector with three 32-channel ADG731 multiplexers permitted electrochemical measurements using single-channel commercial potentiostats. Voltammetric experiments were conducted to demonstrate the reliability, stability, and reproducibility of the FPC array and interfacing hardware. The combination of these devices represents an accessible hardware platform with robust, functionalizable electrodes, a simple connection interface with commercial potentiostats, and a low cost through the use of off-the-shelf components. Our reported strategy holds great promise to facilitate multiplexed electroanalysis in next-generation sensors to increase statistical sample size and multianalyte detection capabilities.

Sirtuin 2 Regulates Protein LactoylLys Modifications.

Post-translational modifications (PTMs) play roles in both physiological and pathophysiological processes through the regulation of enzyme structure and function. We recently identified a novel PTM, lactoylLys, derived through a nonenzymatic mechanism from the glycolytic by-product, lactoylglutathione. Under physiologic scenarios, glyoxalase 2 prevents the accumulation of lactoylglutathione and thus lactoylLys modifications. What dictates the site-specificity and abundance of lactoylLys PTMs, however, remains unknown. Here, we report sirtuin 2 as a lactoylLys eraser. Using chemical biology and CRISPR-Cas9, we show that SIRT2 controls the abundance of this PTM both globally and on chromatin. These results address a major gap in our understanding of how nonenzymatic PTMs are regulated and controlled.

ß-Cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion.

ß-Cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle, and adipose. Previously, we have demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic ß-cells. In this study, we examined whether Nr4a expression impacts pancreatic ß-cell mitochondrial function. Here, we show that ß-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in ß-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16 mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose-stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP and ultimately inhibits glucose-stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for ß-cell mitochondrial function and insulin secretion.

CEBPA Overexpression Enhances ß-Cell Proliferation and Survival.

A commonality between type 1 and type 2 diabetes is the decline in functional ß-cell mass. The transcription factor Nkx6.1 regulates ß-cell development and is integral for proper ß-cell function. We have previously demonstrated that Nkx6.1 depends on c-Fos mediated upregulation and the nuclear hormone receptors Nr4a1 and Nr4a3 to increase ß-cell insulin secretion, survival, and replication. Here, we demonstrate that Nkx6.1 overexpression results in upregulation of the bZip transcription factor CEBPA and that CEBPA expression is independent of c-Fos regulation. In turn, CEBPA overexpression is sufficient to enhance INS-1 832/13 ß-cell and primary rat islet proliferation. CEBPA overexpression also increases the survival of ß-cells treated with thapsigargin. We demonstrate that increased survival in response to ER stress corresponds with changes in expression of various genes involved in the unfolded protein response, including decreased Ire1a expression. These data show that CEBPA is sufficient to enhance functional ß-cell mass by increasing ß-cell proliferation and modulating the unfolded protein response.

In Situ Preconcentration and Quantification of Cu2+ via Chelating Polymer-Wrapped Multiwalled Carbon Nanotubes.

Trace analysis of heavy metals in complex, environmentally relevant matrices remains a significant challenge for electrochemical sensors employing stripping voltammetry-based detection schemes. We present an alternative method capable of selectively preconcentrating Cu2+ ions at the electrode surface using chelating polymer-wrapped multiwalled carbon nanotubes (MWCNTs). An electrochemical sensor consisting of poly-4-vinyl pyridine (P4VP)-wrapped MWCNTs anchored to a poly(ethylene terephthalate) (PET)-modified gold electrode (r = 1.5 mm) was designed, produced, and evaluated. The P4VP is shown to form a strong association with Cu2+ ions, permitting preconcentration adjacent to the electrode surface for interrogation via cyclic voltammetry. The sensor exhibited a detection limit of 0.5 ppm with a linear range of 1.1-13.8 ppm (16.6-216 µM) and a relative standard deviation (RSD) of 4.9% at the Environmental Protection Agency (EPA) limit of 1.3 ppm. Evaluation in tap water, lake water, ocean water, and deionized water rendered similar results, highlighting the generalizability of the presented preconcentration strategy. The advantages of electrochemical analysis paired with polymeric chelation represent an effective platform for the design and deployment of heavy metal sensors for continuous monitoring of natural waters.

Instrumenting Polyodon spathula (Paddlefish) Rostra in Flowing Water with Strain Gages and Accelerometers.

The prominent rostrum of the North American Paddlefish, supported by a lattice-like endoskeleton, is highly durable, making it an important candidate for bio-inspiration studies. Energy dissipation and load-bearing capacity of the structure from extreme physical force has been demonstrated superior to that of man-made systems, but response to continuous hydraulic forces is unknown and requires special instrumentation for in vivo testing on a live fish. A single supply strain gage amplifier circuit has been combined with a digital three-axis accelerometer, implemented in a printed circuit board (PCB), and integrated with the commercial-off-the-shelf Adafruit Feather M0 datalogger with a microSD card. The device is battery powered and enclosed in silicon before attachment around the rostrum with a silicon strap "watch band." As proof-of-concept, we tested the instrumentation on an amputated Paddlefish rostrum in a water-filled swim tunnel and successfully obtained interpretable data. Results indicate that this design could work on live swimming fish in future in vivo experiments.

Common gut microbial metabolites of dietary flavonoids exert potent protective activities in ß-cells and skeletal muscle cells.

Flavonoids are dietary compounds with potential anti-diabetes activities. Many flavonoids have poor bioavailability and thus low circulating concentrations. Unabsorbed flavonoids are metabolized by the gut microbiota to smaller metabolites, which are more bioavailable than their precursors. The activities of these metabolites may be partly responsible for associations between flavonoids and health. However, these activities remain poorly understood. We investigated bioactivities of flavonoid microbial metabolites [hippuric acid (HA), homovanillic acid (HVA), and 5-phenylvaleric acid (5PVA)] in primary skeletal muscle and ß-cells compared to a native flavonoid [(-)-epicatechin, EC]. In muscle, EC was the most potent stimulator of glucose oxidation, while 5PVA and HA simulated glucose metabolism at 25 µM, and all compounds preserved mitochondrial function after insult. However, EC and the metabolites did not uncouple mitochonndrial respiration, with the exception of 5PVA at10 µM. In ß-cells, all metabolites more potently enhanced glucose-stimulated insulin secretion (GSIS) compared to EC. Unlike EC, the metabolites appear to enhance GSIS without enhancing ß-cell mitochondrial respiration or increasing expression of mitochondrial electron transport chain components, and with varying effects on ß-cell insulin content. The present results demonstrate the activities of flavonoid microbial metabolites for preservation of ß-cell function and glucose utilization. Additionally, our data suggest that metabolites and native compounds may act by distinct mechanisms, suggesting complementary and synergistic activities in vivo which warrant further investigation. This raises the intriguing prospect that bioavailability of native dietary flavonoids may not be as critical of a limiting factor to bioactivity as previously thought.

Monomeric cocoa catechins enhance ß-cell function by increasing mitochondrial respiration.

A hallmark of type 2 diabetes (T2D) is ß-cell dysfunction and the eventual loss of functional ß-cell mass. Therefore, mechanisms that improve or preserve ß-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on ß-cell function. INS-1 832/13-derived ß-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract and with oligomeric or polymeric procyanidin-rich fraction demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in ß-cell fuel utilization. Mitochondrial complex III, IV and V components are up-regulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with nuclear factor, erythroid 2 like 2 (Nrf2) nuclear localization and expression of Nrf2 target genes including nuclear respiratory factor 1 (Nrf1) and GA binding protein transcription factor alpha subunit (GABPA), essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and up-regulation of genes critical for mitochondrial respiration, glucose-stimulated insulin secretion and ultimately improved ß-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

Nkx6.1-mediated insulin secretion and ß-cell proliferation is dependent on upregulation of c-Fos.

Understanding the molecular pathways that enhance ß-cell proliferation, survival, and insulin secretion may be useful to improve treatments for diabetes. Nkx6.1 induces proliferation through the Nr4a nuclear receptors, and improves insulin secretion and survival through the peptide hormone VGF. Here we demonstrate that Nkx6.1-mediated upregulation of Nr4a1, Nr4a3, and VGF is dependent on c-Fos expression. c-Fos overexpression results in activation of Nkx6.1 responsive genes and increases ß-cell proliferation, insulin secretion, and cellular survival. c-Fos knockdown impedes Nkx6.1-mediated ß-cell proliferation and insulin secretion. These data demonstrate that c-Fos is critical for Nkx6.1-mediated expansion of functional ß-cell mass.