UTRs and Ago-2/miR-335 Complex Restricts Amylin Translation in Insulinoma and Human Pancreatic ß-Cells.

Amylin promoter and transcriptional factors are well-established, inducible factors in the production of the main amyloidogenic pancreatic hormone, human islet amyloid peptide (hIAPP) or amylin. However, posttranscriptional mechanisms driving hIAPP expression in pancreas remain enigmatic, and hence were explored here. The translational assay revealed that both 5' and 3' untranslated regions (UTRs) of hIAPP restricted expression of the luciferase constructs only in constructs driven by the hIAPP promoter. Bioinformatics analysis revealed several putative seed sequences for a dozen micro RNAs (miRNAs) in hIAPP's 3' UTR. miR-182, miR-335, and miR-495 were the most downregulated miRNAs in stressed human islets exposed to endoplasmic reticulum (ER) or metabolic stressors, thapsigargin (TG) or high glucose (HG). Correspondingly, miR-335 mimics alone or in combination with miR-495 and miR-182 mimics significantly and potently (>3-fold) reduced hIAPP protein expression in HG-treated cultured human islets. siRNA-mediated silencing of Ago2 but not Ago1 significantly stimulated hIAPP expression and secretion from transfected, HG-treated human islets. Conversely, ectopic expression of Ago2 in hIAPP-expressing RIN-m5F cell line driven by CMV promoter reduced hIAPP intracellular protein levels. Collectively, the results point to a novel and synergistic role for hIAPP promoter, 5/3' UTRs and Ago-2/miR-335 complex in post-transcriptional regulation of hIAPP gene expression in normal and metabolically active ß-cells.

Analyzing Gene Expression After Administration of Low-Intensity Therapeutic Ultrasound in Human Islet Cells.

OBJECTIVES: Diabetes mellitus is a complex heterogenous metabolic disease that significantly affects the world population. Although many treatments exist, including medications such as metformin, sulfonylureas, and glucagon-like peptide-1 (GLP) receptor agonist, there is growing interest in finding alternative methods to noninvasively treat this disease. It has been previously shown that low-intensity ultrasound stimulation of pancreatic ß-cells in mice can elicit insulin secretion as a potential treatment for this disease. This is desirable as therapeutic ultrasound has the ability to induce bioeffects while selectively focusing deep within tissues, allowing for modulation of hormone secretion in the pancreas to mitigate insufficient levels of insulin. METHODS: Exactly 800 kHz ultrasound with intensity 0.5 W/cm2 was administered 5 minutes continuously, that is, 100% duty cycle, to donor pancreatic human islets, followed by 1 hour incubation and RT-qPCR to assess the effect of ultrasound stimulation on gene expression. The genes were insulin (INS), glucagon (Glu), amylin (Amy), and binding immunoglobulin protein (BiP). Nine donor pancreatic human islets were used to assess insulin and glucagon secretion, while eight samples were used for amylin and BiP. Fold change (FC) was calculated to analyze the effect of ultrasound stimulation on the gene expression of the donor islet cells. High-glucose and thapsigargin-treated islets were utilized as positive controls. Cell viability testing was done using a Trypan Blue Exclusion Test. RESULTS: Ultrasound stimulation did not cause a statistically significant upregulation in any of the tested genes (INS FC = 1.15, P-value = .5692; Glu FC = 1.60, P-value = .2231; Amy FC, P-value = .2863; BiP FC = 2.68, P-value = .3907). CONCLUSIONS: The results of this study show that the proposed ultrasound treatment parameters do not appear to significantly affect gene expression of any gene tested.

Therapeutic Ultrasound Effects on Human Induced Pluripotent Stem Cell Cardiomyocytes Measured Optically and with Spectral Ultrasound.

To the best of our knowledge, therapeutic ultrasound (TUS) is thus far an unexplored means of delivering mechanical stimulation to cardiomyocyte cultures, which is necessary to engineer a more mature cardiomyocyte phenotype in vitro. Spectral ultrasound (SUS) may provide a way to non-invasively, non-disruptively and inexpensively monitor growth and change in cell cultures over long periods. Compared with other measurement methods, SUS as an acoustic measurement tool will not be affected by an acoustic therapy, unlike electrical measurement methods, in which motion caused by acoustic therapy can affect measurements. Further SUS has the potential to provide functional as well as morphological information in cell cultures. Human induced pluripotent stem cell cardiomyocytes (iPS-CMs) were imaged with calcium fluorescence microscopy while TUS was being applied. TUS was applied at 600 kHz and 1, 3.4 and 6 W/cm2 for a continuous 1 s pulse. Measures of the instantaneous beat frequency, repolarization rate and calcium spike amplitude were calculated from the fluorescence data. At 600 kHz, TUS at 1 and 6 W/cm2 had significant effects on the shortening of both the repolarization rate and instantaneous beat rate of the iPS-CMs (p < 0.05), while TUS at 3.4 and 6 W/cm2 had significant effects on the shortening of the calcium spike amplitude (p < 0.05). Three SUS measures and one gray-level measure were captured from the iPS-CM monolayers while they were simultaneously being imaged with calcium-labeled confocal microscopy. The gray-level measure performed the best of all SUS measures; however, it was not reliable enough to produce a consistent determination of the beat rate of the cell. Finally, SUS measures were captured using three different transducers while simultaneously applying TUS. A center-of-mass (COM) measure calculated from the wavelet transform scalogram of the time-averaged radiofrequency data revealed that SUS was able to detect a change in the frequency content of the reflected ultrasound at 1 and 6 W/cm2 before and after ultrasound application (p < 0.05), showing promise for the ability of SUS to measure changes in the beating behavior of iPS-CMs. Overall, SUS is promising as a method for constant monitoring of dynamic cell and tissue culture and growth.

Molecular Mechanisms of Amylin Turnover, Misfolding and Toxicity in the Pancreas.

Amyloidosis is a common pathological event in which proteins self-assemble into misfolded soluble and insoluble molecular forms, oligomers and fibrils that are often toxic to cells. Notably, aggregation-prone human islet amyloid polypeptide (hIAPP), or amylin, is a pancreatic hormone linked to islet ß-cells demise in diabetics. The unifying mechanism by which amyloid proteins, including hIAPP, aggregate and kill cells is still matter of debate. The pathology of type-2 diabetes mellitus (T2DM) is characterized by extracellular and intracellular accumulation of toxic hIAPP species, soluble oligomers and insoluble fibrils in pancreatic human islets, eventually leading to loss of ß-cell mass. This review focuses on molecular, biochemical and cell-biology studies exploring molecular mechanisms of hIAPP synthesis, trafficking and degradation in the pancreas. In addition to hIAPP turnover, the dynamics and the mechanisms of IAPP-membrane interactions; hIAPP aggregation and toxicity in vitro and in situ; and the regulatory role of diabetic factors, such as lipids and cholesterol, in these processes are also discussed.

FoxA2 and RNA Pol II mediate human islet amyloid polypeptide turnover in ER-stressed pancreatic ß-cells.

Here, we investigated transcriptional and trafficking mechanisms of human islet amyloid polypeptide (hIAPP) in normal and stressed ß-cells. In high glucose-challenged human islets and rat insulinoma cells overexpressing hIAPP, cell fractionation studies revealed increased accumulation of hIAPP. Unexpectedly, a significant fraction (up to 22%) of hIAPP was found in the nuclear soluble and chromatin-enriched fractions of cultured human islet and rat insulinoma cells. The nucleolar accumulation of monomeric forms of hIAPP did not have any adverse effect on the proliferation of ß-cells nor did it affect nucleolar organization or function. However, intact nucleolar organization and function were essential for hIAPP expression under normal and ER-stress conditions as RNA polymerase II inhibitor, α-amanitin, reduced hIAPP protein expression evoked by high glucose and thapsigargin. Promoter activity studies revealed the essential role of transcription factor FoxA2 in hIAPP promoter activation in ER-stressed ß-cells. Transcriptome and secretory studies demonstrate that the biosynthetic and secretory capacity of islet ß-cells was preserved during ER stress. Thus, the main reason for increased intracellular hIAPP accumulation is its enhanced biosynthesis under these adverse conditions.

Effect of Therapeutic Ultrasound on the Release of Insulin, Glucagon, and Alpha-Amylase from Ex Vivo Pancreatic Models.

OBJECTIVES: Our previously published studies showed the potential of therapeutic ultrasound (US) as a novel non-pharmacological alternative for the treatment of secretory deficiencies in type 2 diabetes. Despite showing enhanced insulin release from beta cells, these studies did not explore the potential effects of US treatment on other cells in the islets of Langerhans such as glucagon-secreting alpha cells or acinar cells of the exocrine pancreas. METHODS: We applied US parameters found capable of safely stimulating insulin secretion from pancreatic beta cells (f = 800 kHz, ISPTA  = 0.5-1 W/cm2 , 5 minutes) to a diced rabbit pancreas model in culture plates (n = 6 per group). Released quantities of insulin and glucagon in response to US treatment were measured by collecting aliquots of the extracellular medium prior to the start of the treatment (t = 0 minute), immediately after treatment (t = 5 minutes) and 30 minutes after the end of treatment (t = 35 minutes). Potential release of digestive enzyme alpha-amylase as a result of US treatment was evaluated in rabbit pancreas experiments. Preliminary studies were also performed in a small number of human pancreatic islets in culture plates (n = 3 per group). The general integrity of the US-treated rabbit pancreatic tissue and human pancreatic islets was evaluated through histological analysis. RESULTS: While sham-treated rabbit pancreas samples showed decreased extracellular insulin content, there was an increase in insulin release at t = 5 minutes from samples treated with US at 800 kHz and 1 W/cm2 (P <.005). Furthermore, no further insulin release was detected at t = 35 minutes. No statistically significant difference in extracellular glucagon and alpha-amylase concentrations was observed between US-treated and sham rabbit pancreas groups. Preliminary studies in human islets appeared to follow trends observed in rabbit pancreas studies. Islet and other pancreatic tissue integrity did not appear to be affected by the US treatment. CONCLUSION: A potential US-based strategy for enhanced insulin release would require optimization of insulin secretion from pancreatic beta cells while minimizing glucagon and pancreatic enzyme secretions.

Ex Vivo Imaging of Ultrasound-Stimulated Metabolic Activity in Rat Pancreatic Slices.

Ultrasound has previously been reported to produce a reversible stimulatory effect in cultured rat beta cells. Here, we quantified and assessed dynamic metabolic changes in an in situ pancreatic slice model evoked by ultrasound application. After plating, pancreas slices were imaged using a confocal microscope at 488 and 633 nm to image lipodamine dehydrogenase (Lip-DH) autofluorescence and a far red fluorescence, respectively. Ultrasound was applied at intensities of 0.5 and 1 W/cm2 at both 800 kHz and 1 MHz. Additionally, 800 kHz at 1 W/cm2 was applied in a pulsing scheme. No ultrasound (control) and glucose application experiments were performed. A difference in fluorescence signal before and after treatment application was the metric for analysis. Comparison of experimental groups using far red fluorescence revealed significant differences between all experimental groups and control in the islet (p < 0.05) and between all ultrasound experimental groups and control (p < 0.05) in pancreatic exocrine tissue. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed using Lip-DH autofluorescence that glucose produces a significantly increased metabolic response in islet tissue compared with exocrine tissue (p < 0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner compared with continuous ultrasound application. Our results indicate that therapeutic ultrasound may have a stimulatory metabolic effect on the pancreatic islets similar to that of glucose.

Non-Segmented ECG bio-identification using Short Time Fourier Transform and Fréchet Mean Distance.

In the recent years, the Electrocardiogram (ECG) based biometric identification has been a subject of considerable research interest. In this paper, we present non-fiducial method for ECG-identification using the short time Fourier transform (STFT), and Frechet mean distance-based algorithms to find the similarity between the STFTs of different people. In this study, we select randomly the training and test data of the ECG in order to test the stability of the method. We apply our proposed method on 124 ECG records of 62 subjects from the publicly available ECG ID database from physionet website. Our preliminary results indicate that the Frechet mean based ECG identification has 96.45% average identification accuracy and therefore can be potentially useful in various applications.

Therapeutic Ultrasound-Induced Insulin Release in Vivo.

The tolerability and efficacy of low-frequency, low-intensity therapeutic ultrasound-induced insulin release was investigated in a pre-clinical in vivo murine model. The treatment groups received a single 5-min continuous sonication at 1 MHz and 1.0 W/cm2. Insulin and glucagon levels in the serum were determined using enzyme-linked immunosorbent assay. The pancreas was excised and sectioned for histologic analysis. In terminal studies, we observed a moderate (∼50 pM) but significant increase in blood insulin concentration in vivo immediately after sonication compared with a decrease of approximately 60 pM in sham animals (n < 6, p < 0.005). No difference was observed in the change in glucose or glucagon concentrations between groups. Comparisons of hematoxylin and eosin-stained terminal and survival pancreatic tissue revealed no visible differences or evidence of damage. This study is the first step in assessing the translational potential of therapeutic ultrasound as a treatment for early stages of type 2 diabetes.

An inducible model of human amylin overexpression reveals diverse transcriptional changes.

Human Islet Amyloid Polypeptide or amylin is a neuroendocrine peptide with important endocrine and paracrine functions. Excessive production and accumulation of human amylin in the pancreas can lead to its aggregation and apoptosis of islet ß-cells. Amylin has been shown to function within the central nervous system to decrease food intake, and more recently, it has been revealed that amylin is directly transcribed from neurons of the central nervous system, including the hypothalamus, arcuate nucleus, medial preoptic area, and nucleus accumbens. These findings alter the current model of how amylin targets the nervous system, and as a result may lead to obesity and type II diabetes mellitus. Here we set out to use Caenorhabditis elegans as an inducible in vivo model system to study the effects of amylin overexpression in tissues that include the nervous system. We profiled the transcriptional changes in transgenic animals expressing human amylin through RNA-seq. Using this genome-wide approach our results revealed for the first time that expression of human amylin in tissues including the nervous system induce diverse physiological responses in various signaling pathways. From our characterization of transgenic C. elegans animals expressing human amylin, we also observed specific defects in neural developmental programs as well as sensory behavior. Taken together, our data demonstrate the utility of using C. elegans as a valuable in vivo model to study human amylin toxicity.

Ultrasound-Induced Insulin Release as a Potential Novel Treatmentfor Type 2 Diabetes Mellitus.

Therapeutic ultrasound presents a potential novel treatment for type 2 diabetes mellitus that utilizes the non-invasive application of ultrasound energy to treat secretory defects in the earlier stages of the disease. Our previous studies have shown that ultrasound is capable of stimulating insulin release from pancreatic beta cells, safely and effectively. This study aims to both examine the calcium-dependent mechanisms of ultrasound-mediated insulin release from pancreatic beta cells using three complementary modalities - carbon fiber amperometry, ELISA studies, and Ca2+ fluorescence imaging - and to study the translational potential of therapeutic ultrasound using transgenic hyperglycemic mice for safety and efficacy studies.

Functional proteasome complex is required for turnover of islet amyloid polypeptide in pancreatic ß-cells.

Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed ß-cells evoked by chronic hyperglycemia. Moderate inhibition (10-35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed ß-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma ß-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in ß-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.

Apoptosis signal regulating kinase-1 and NADPH oxidase mediate human amylin evoked redox stress and apoptosis in pancreatic beta-cells.

Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet ß-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to ß-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic ß-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. ß-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in ß-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic ß-cells.

The Molecular Physiopathogenesis of Islet Amyloidosis.

Human islet amyloid polypeptide or amylin (hA) is a 37-amino acid peptide hormone produced and co-secreted with insulin by pancreatic ß-cells. Under physiological conditions, hA regulates a broad range of biological processes including insulin release and slowing of gastric emptying, thereby maintaining glucose homeostasis. However, under the pathological conditions associated with type 2 diabetes mellitus (T2DM), hA undergoes a conformational transition from soluble random coil monomers to alpha-helical oligomers and insoluble ß-sheet amyloid fibrils or amyloid plaques. There is a positive correlation between hA oligomerization/aggregation, hA toxicity, and diabetes progression. Because the homeostatic balance between hA synthesis, release, and uptake is lost in diabetics and hA aggregation is a hallmark of T2DM, this chapter focuses on the biophysical and cell biology studies investigating molecular mechanisms of hA uptake, trafficking, and degradation in pancreatic cells and its relevance to h's toxicity. We will also discuss the regulatory role of endocytosis and proteolytic pathways in clearance of toxic hA species. Finally, we will discuss potential pharmacological approaches for specific targeting of hA trafficking pathways and toxicity in islet ß-cells as potential new avenues toward treatments of T2DM patients.

Calcium-dependent ultrasound stimulation of secretory events from pancreatic beta cells.

BACKGROUND: Our previous studies have indicated that ultrasound can stimulate the release of insulin from pancreatic beta cells, providing a potential novel treatment for type 2 diabetes. The purpose of this study was to explore the temporal dynamics and Ca2+-dependency of ultrasound-stimulated secretory events from dopamine-loaded pancreatic beta cells in an in vitro setup. METHODS: Carbon fiber amperometry was used to detect secretion from INS-1832/13 beta cells in real time. The levels of released insulin were also measured in response to ultrasound treatment using insulin-specific ELISA kit. Beta cells were exposed to continuous wave 800 kHz ultrasound at intensities of 0.1 W/cm2, 0.5 W/cm2 and 1 W/cm2 for several seconds. Cell viability tests were done with trypan blue dye exclusion test and MTT analysis. RESULTS: Carbon fiber amperometry experiments showed that application of 800 kHz ultrasound at intensities of 0.5 and 1 W/cm2 was capable of stimulating secretory events for durations lasting as long as the duration of the stimulus. Furthermore, the amplitude of the detected peaks was reduced by 64% (p < 0.01) when extracellular Ca2+ was chelated with 10 mM EGTA in cells exposed to ultrasound intensity of 0.5 W/cm2. Measurements of released insulin in response to ultrasound stimulation showed complete inhibition of insulin secretion by chelating extracellular Ca2+ with 10 mM EGTA (p < 0.01). Viability studies showed that 800 kHz, 0.5 W/cm2 ultrasound did not cause any significant effects on viability and metabolic activity in cells exposed to ultrasound as compared to sham-treated cells. CONCLUSIONS: Our results demonstrated that application of ultrasound was capable of stimulating the release of insulin from pancreatic beta cells in a safe, controlled and Ca2+-dependent manner.

Ultrasound Stimulation of Insulin Release from Pancreatic Beta Cells as a Potential Novel Treatment for Type 2 Diabetes.

Type 2 diabetes mellitus is a complex metabolic disease that has reached epidemic proportions in the United States and around the world. This disease is characterized by loss of insulin secretion and, eventually, destruction of insulin-producing pancreatic beta cells. Controlling type 2 diabetes is often difficult as pharmacological management routinely requires complex therapy with multiple medications, and loses its effectiveness over time. The objective of this study was to explore the effectiveness of a novel, non-pharmacological approach that uses the application of ultrasound energy to augment insulin release from rat INS 832/13 beta cells. The cells were exposed to unfocused ultrasound for 5 min at a peak intensity of 1 W/cm2 and frequencies of 400 kHz, 600 kHz, 800 kHz and 1 MHz. Insulin release was measured with enzyme-linked immunosorbent assay and cell viability was assessed via the trypan blue dye exclusion test. A marked release (approximately 150 ng/106 cells, p < 0.05) of insulin was observed when beta cells were exposed to ultrasound at 400 and 600 kHz as compared with their initial control values; however, this release was accompanied by a substantial loss in cell viability. Ultrasound application at frequencies of 800 kHz resulted in 24 ng/106 cells released insulin (p < 0.05) as compared with its unstimulated base level, while retaining cell viability. Insulin release from beta cells caused by application of 800-kHz ultrasound was comparable to that reported by the secretagogue glucose, thus operating within physiological secretory capacity of these cells. Ultrasound has potential as a novel and alternative method to current approaches aimed at correcting secretory deficiencies in patients with type 2 diabetes.

Proteasome regulates turnover of toxic human amylin in pancreatic cells.

Toxic human amylin (hA) oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Although recent studies demonstrated a causal connection between hA uptake and toxicity in pancreatic cells, the mechanism of amylin's clearance following its internalization and its relationship to toxicity is yet to be determined, and hence was investigated here. Using pancreatic rat insulinoma ß-cells and human islets as model systems, we show that hA, following its internalization, first accumulates in the cytosol followed by its translocation into nucleus, and to a lesser extent lysosomes, keeping the net cytosolic amylin content low. An increase in hA accumulation in the nucleus of pancreatic cells correlated with its cytotoxicity, suggesting that its excessive accumulation in the nucleus is detrimental. hA interacted with 20S core and 19S lid subunits of the ß-cell proteasomal complex, as suggested by immunoprecipitation and confocal microscopy studies, which subsequently resulted in a decrease in the proteasome's proteolytic activity in these cells. In vitro binding and activity assays confirmed an intrinsic and potent ability of amylin to interact with the 20S core complex thereby modulating its proteolytic activity. Interestingly, less toxic and aggregation incapable rat amylin (rA) showed a comparable inhibitory effect on proteasome activity and protein ubiquitination, decoupling amylin aggregation/ toxicity and amylin-induced protein stress. In agreement with these studies, inhibition of proteasomal proteolytic activity significantly increased intracellular amylin content and toxicity. Taken together, our results suggest a pivotal role of proteasomes in amylin's turnover and detoxification in pancreatic cells.

Rapid assessment of human amylin aggregation and its inhibition by copper(II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation.

Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin-copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the ß-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the -HSSNN- residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of ß-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of ß-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin-copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors of amyloid protein aggregation.

Role of Cholesterol and Phospholipids in Amylin Misfolding, Aggregation and Etiology of Islet Amyloidosis.

Amyloidosis is a biological event in which proteins undergo structural transitions from soluble monomers and oligomers to insoluble fibrillar aggregates that are often toxic to cells. Exactly how amyloid proteins, such as the pancreatic hormone amylin, aggregate and kill cells is still unclear. Islet amyloid polypeptide, or amylin, is a recently discovered hormone that is stored and co-released with insulin from pancreatic islet ß-cells. The pathology of type 2 diabetes mellitus (T2DM) is characterized by an excessive extracellular and intracellular accumulation of toxic amylin species, soluble oligomers and insoluble fibrils, in islets, eventually leading to ß-cell loss. Obesity and elevated serum cholesterol levels are additional risk factors implicated in the development of T2DM. Because the homeostatic balance between cholesterol synthesis and uptake is lost in diabetics, and amylin aggregation is a hallmark of T2DM, this chapter focuses on the biophysical and cell biology studies exploring molecular mechanisms by which cholesterol and phospholipids modulate secondary structure, folding and aggregation of human amylin and other amyloid proteins on membranes and in cells. Amylin turnover and toxicity in pancreatic cells and the regulatory role of cholesterol in these processes are also discussed.

Detecting breast cancer using microwave imaging and stochastic optimization.

Breast cancer detection is one of the most important problems in health care as it is second most frequent cancer according to WHO. Breast cancer is among cancers which are most probably curable, only if it is diagnosed at early stages. To this purpose it has been recently proposed that microwave imaging could be used as a cheaper and safer alternative to the commonly used combination of mammography. From a physical standpoint breast cancer can be modelled as a scatterer with a significantly (tenfold) larger conductivity than a healthy tissue. In our previous work we proposed a maximum likelihood based method for detection of cancer which estimates the unknown parameters by minimizing the residual error vector assuming that the error can be modelled as a multivariate (multiple antennas) random variable. In this paper we utilize stochastic optimization technique and evaluate its applicability to the detection of cancer using numerical models. Although these models have significant limitations they are potentially useful as they provide insight in required levels of noise in order to achieve desirable detection rates.