Leveraging Ion-Ion and Ion-Photon Activation to Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin Case Study.

Gas-phase sequencing of large intact proteins (>30 kDa) via tandem mass spectrometry is an inherently challenging process that is further complicated by the extensive overlap of multiply charged product ion peaks, often characterized by a low signal-to-noise ratio. Disulfide bonds exacerbate this issue because of the need to cleave both the S-S and backbone bonds to liberate sequence informative fragments. Although electron-based ion activation techniques such as electron transfer dissociation (ETD) have been proven to rupture disulfide bonds in whole protein ions, they still struggle to produce extensive sequencing when multiple, concatenated S-S bonds are present on the same large polypeptide chain.

NMDA Receptors in POMC Neurons Connect Exercise With Insulin Sensitivity.

Increased arcuate proopiomelanocortin (POMC) neuron activity improves glucose metabolism and reduces appetite, facilitating weight loss. We recently showed that arcuate POMC neurons are activated by exercise. However, the role of excitatory glutamatergic input in these neurons and the metabolic outcomes of exercise remains undefined.

Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction-Orbitrap mass spectrometry.

Modern mass spectrometry technology allows for extensive sequencing of the ~ 25 kDa subunits of monoclonal antibodies (mAbs) produced by IdeS proteolysis followed by disulfide bond reduction, an approach known as middle-down mass spectrometry (MD MS). However, the spectral congestion of tandem mass spectra of large polypeptides dramatically complicates fragment ion assignment. Here, we report the development and benchmark of an MD MS strategy based on the combination of different ion fragmentation techniques with proton transfer charge reduction (PTCR) to simplify the gas-phase sequencing of mAb subunits.

Upregulation of Xbp1 in NPY/AgRP neurons reverses diet-induced obesity and ameliorates leptin and insulin resistance.

The molecular mechanisms underlying neuronal leptin and insulin resistance in obesity and diabetes are not fully understood. In this study, we show that induction of the unfolded protein response transcription factor, spliced X-box binding protein 1 (Xbp1s), in Agouti-Related Peptide (AgRP) neurons alone, is sufficient to not only protect against but also significantly reverse diet-induced obesity (DIO) as well as improve leptin and insulin sensitivity, despite activation of endoplasmic reticulum stress. We also demonstrate that constitutive expression of Xbp1s in AgRP neurons contributes to improved insulin sensitivity and glucose tolerance.

Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry.

Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e.

Time and metabolic state-dependent effects of GLP-1R agonists on NPY/AgRP and POMC neuronal activity in vivo.

Objective: Long-acting glucagon-like peptide-1 receptor agonists (GLP-1RAs), like liraglutide and semaglutide, are viable treatments for diabetes and obesity. Liraglutide directly activates hypothalamic proopiomelanocortin (POMC) neurons while indirectly inhibiting Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons ex vivo. While temporal control of GLP-1R agonist concentration as well as accessibility to tissues/cells can be achieved with relative ease ex vivo, in vivo this is dependent upon the pharmacokinetics of these agonists and relative penetration into structures of interest.

PERK in POMC neurons connects celastrol with metabolism.

ER stress and activation of the unfolded protein response in the periphery as well as the central nervous system have been linked to various metabolic abnormalities. Chemically lowering protein kinase R-like ER kinase (PERK) activity within the hypothalamus leads to decreased food intake and body weight. However, the cell populations required in this response remain undefined.

Glucagon-like peptide-1 (GLP-1) signalling in the brain: From neural circuits and metabolism to therapeutics.

Glucagon-like-peptide-1 (GLP-1) derived from gut enteroendocrine cells and a discrete population of neurons in the caudal medulla acts through humoral and neural pathways to regulate satiety, gastric motility and pancreatic endocrine function. These physiological attributes contribute to GLP-1 having a potent therapeutic action in glycaemic regulation and chronic weight management. In this review, we provide an overview of the neural circuits targeted by endogenous versus exogenous GLP-1 and related drugs.

Effects of metabolic state on the regulation of melanocortin circuits.

Dysfunction in neurophysiological systems that regulate food intake and metabolism are at least partly responsible for obesity and related comorbidities. An important component of this process is the hypothalamic melanocortin system, where an imbalance can result in severe obesity and deficits in glucose metabolism. Exercise offers many health benefits related to cardiovascular improvements, hunger control, and blood glucose homeostasis.

Direct and indirect effects of liraglutide on hypothalamic POMC and NPY/AgRP neurons - Implications for energy balance and glucose control.

Objective: The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, stimulates insulin secretion and efficiently suppresses food intake to reduce body weight. As such, liraglutide is growing in popularity in the treatment of diabetes and chronic weight management. Within the brain, liraglutide has been shown to alter the activity of hypothalamic proopiomelanocortin (POMC) and Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons.

Acute effects of zinc and insulin on arcuate anorexigenic proopiomelanocortin neurons.

Background And Purpose: Acute insulin administration hyperpolarized, with concomitant decrease of firing rate, a subpopulation of arcuate proopiomelanocortin (POMC) and neuropeptide Y/agouti-related peptide cells. This rapid effect on cellular activity has been proposed as a cellular correlate of insulin effects on energy balance and glucose homoeostasis. Recent evidence suggests that zinc in mammalian insulin formulations is required for the insulin-induced inhibition of arcuate POMC neurons, while guinea pig insulin, which fails to bind zinc, activates POMC neurons in mice.

Cellular and synaptic reorganization of arcuate NPY/AgRP and POMC neurons after exercise.

Objective: Hypothalamic Pro-opiomelanocortin (POMC) and Neuropeptide Y/Agouti-Related Peptide (NPY/AgRP) neurons are critical nodes of a circuit within the brain that sense key metabolic cues as well as regulate metabolism. Importantly, these neurons retain an innate ability to rapidly reorganize synaptic inputs and electrophysiological properties in response to metabolic state. While the cellular properties of these neurons have been investigated in the context of obesity, much less is known about the effects of exercise training.

Phosphoinositide 3-Kinase Is Integral for the Acute Activity of Leptin and Insulin in Male Arcuate NPY/AgRP Neurons.

Neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus are part of a neuroendocrine feedback loop that regulates feeding behavior and glucose homeostasis. NPY/AgRP neurons sense peripheral signals (including the hormones leptin, insulin, and ghrelin) and integrate those signals with inputs from other brain regions. These inputs modify both long-term changes in gene transcription and acute changes in the electrical activity of these neurons, leading to a coordinated response to maintain energy and glucose homeostasis.

Substantial Targeting Advantage Achieved by Pulmonary Administration of Colistin Methanesulfonate in a Large-Animal Model.

Colistin, administered as its inactive prodrug colistin methanesulfonate (CMS), is often used in multidrug-resistant Gram-negative pulmonary infections. The CMS and colistin pharmacokinetics in plasma and epithelial lining fluid (ELF) following intravenous and pulmonary dosing have not been evaluated in a large-animal model with pulmonary architecture similar to that of humans. Six merino sheep (34 to 43 kg body weight) received an intravenous or pulmonary dose of 4 to 8 mg/kg CMS (sodium) or 2 to 3 mg/kg colistin (sulfate) in a 4-way crossover study.

Predicting the composition of red wine blends using an array of multicomponent Peptide-based sensors.

Differential sensing using synthetic receptors as mimics of the mammalian senses of taste and smell is a powerful approach for the analysis of complex mixtures. Herein, we report on the effectiveness of a cross-reactive, supramolecular, peptide-based sensing array in differentiating and predicting the composition of red wine blends. Fifteen blends of Cabernet Sauvignon, Merlot and Cabernet Franc, in addition to the mono varietals, were used in this investigation.

Application of ultrasound in grape mash treatment in juice processing.

Recently, application of ultrasound has attracted considerable interest as an alternative approach to traditional methods. In this study, response surface methodology (RSM) was used to optimize the conditions for grape mash treatment by ultrasound and by combination of ultrasound and enzyme. The results indicated that optimal conditions were the temperature of 74 degrees C and the time of 13 min for sonication treatment; and were the enzyme concentration of 0.

Dimension reduction for classification with gene expression microarray data.

An important application of gene expression microarray data is classification of biological samples or prediction of clinical and other outcomes. One necessary part of multivariate statistical analysis in such applications is dimension reduction. This paper provides a comparison study of three dimension reduction techniques, namely partial least squares (PLS), sliced inverse regression (SIR) and principal component analysis (PCA), and evaluates the relative performance of classification procedures incorporating those methods.

Blood glucose distribution and prevalence of diabetes in Hanoi (Vietnam).

Few epidemiologic surveys have been performed to assess the prevalence of diabetes in representative samples, and few data are available on the epidemiologic features of diabetes in Southeast Asia. We report the results of a 1990 study performed in the Hanoi area (Vietnam) on 4,912 subjects (95.0% of the eligible population), aged 15 years or over, selected by a stratified random cluster procedure using the 1989 census list.

Does follicle-stimulating hormone or pregnant mare serum gonadotrophin influence testicular blood flow in rats?

Treatment of adult rats with 25 iu follicle-stimulating hormone (FSH) did not influence testicular blood flow, serum testosterone, vasomotion or intravascular leucocyte concentration at 6, 12 and 24 h after treatment. Treatment of adult rats with 50 iu pregnant mare serum gonadotrophin (PMSG) resulted in a two-fold increase in testicular blood flow at 24 h, and an increase in interstitial fluid volume at 36 h after treatment. This PMSG-induced increase in blood flow did not occur in Leydig cell-depleted animals, suggesting that the effect on blood flow is mediated via the Leydig cells.