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Supplementary Table 4 from Phosphoproteomic Screen Identifies Potential Therapeutic Targets in Melanoma

Supplementary Table Legends 1-6, Figure Legend 1 from Phosphoproteomic Screen Identifies Potential Therapeutic Targets in Melanoma

Supplementary Tables 1-3 from Phosphoproteomic Screen Identifies Potential Therapeutic Targets in Melanoma

Supplementary Table 5 from Phosphoproteomic Screen Identifies Potential Therapeutic Targets in Melanoma

Supplementary Figure 1 from Phosphoproteomic Screen Identifies Potential Therapeutic Targets in Melanoma

Supplementary Table 6 from Phosphoproteomic Screen Identifies Potential Therapeutic Targets in Melanoma

Stevens Johnson syndrome (SJS), a life threatening mucocutaneous disease can present with many variations and rapidly worsen in a short period of time. Various etiologies are associated with these conditions, with adverse drug reaction being the most common. While the reaction itself is self-limited, little is known about the frequency and risk factors associated with long term SJS sequelae. Presenting a case of a 40 years old male patient who presented to our dental OPD with the chief complaint of burning sensation in the mouth with complete loss of eyesight, all these resultant due to treatment of Stevens Johnson Syndrome. Therapeutic management was done after proper investigations. This case emphasizes the importance of recognition of long term sequelae of Stevens Johnson syndrome as this is a rare but serious condition that must be diagnosed and treated appropriately. Clinicians should be aware of the potential for long term complications among Stevens Johnson survivors.

In this paper, we consider a novel underlay relayed non-orthogonal multiple access (NOMA) network with cooperative relaying by an energy harvesting (EH) near-user ( $NU$ ) so to improve the performance of a far-user ( $FU$ ) without requiring the $NU$ to expend its own battery energy. The $NU$ is assumed to harvest energy from the relay using the power-splitting protocol. Expressions are derived for end-to-end outage probability and throughput of both $NU$ and $FU$ . Although the source, relay and $NU$ limit their transmit powers to ensure that the peak interference caused to the primary is below the interference temperature limit (ITL), the network still performs better than orthogonal multiple access (OMA) based network. We show that combining of signals from relay and $NU$ ensures good $FU$ performance in this power constrained scenario. We demonstrate that both NOMA power allocation parameter as well as the energy harvesting parameter need to be chosen carefully to maximize the sum throughput. Simulation results corroborate the derived analytical expressions.

Objective Non-alcoholic fatty liver (NAFL) associated with obesity is a major cause of liver diseases which can progress to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) plays an important role in liver metabolism and is also a potential marker for NAFL. Here we aimed to test the effect of FGF21 deficiency on liver pathology in mice consuming a conventional high fat, high sucrose (HFHS) obesogenic diet for up to 52 weeks. Methods C57BL6 WT and FGF21 KO mice were fed a conventional obesogenic diet and were evaluated at 16 and 52 weeks. Evaluation included metabolic assessment, liver pathology, and transcriptomic analysis. Results With consumption of HFHS diet, FGF21 deficient mice (FGF21 KO) develop excess fatty liver within 16 weeks. Hepatic pathology progresses and at 52 weeks FGF21 KO mice show significantly worse fibrosis and 78% of mice develop HCC; in contrast only 6% of WT mice develop HCC. Well differentiated hepatocellular carcinomas in FGF21 KO mice were characterized by expanded hepatic plates, loss of reticulin network, cytologic atypia, and positive immunostaining for glutamine synthetase. Microarray analysis reveals enrichment of several fibroblast growth factor signaling pathways in the tumors. Conclusions In addition to attenuating inflammation and fibrosis in mice under a number of dietary challenges, we show here that FGF21 is required to limit the progression from NAFL to HCC in response to prolonged exposure to an obesogenic diet. The induction of hepatic FGF21 in response to the high fat, high sucrose obesogenic diet may play an important role in limiting progression of liver pathology from NAFL to HCC., Highlights • FGF21 deficiency in the context of a standard high, fat high sucrose, obesogenic diet increases liver pathology with rapid progression to NASH. • Hepatic pathology in FGF21 KO mice progresses to hepatocellular carcinoma. • Microarray analysis of the tumors reveals enrichment of several fibroblast growth factor signaling pathways. • Results noted here are similar to increased rates of progressive NAFLD in obese populations consuming Western style diets.

Metabolic diseases such as diabetes and obesity are a growing healthcare concern, and their increasing rates are attributed to increased consumption of carbohydrate-rich diets and sugar-sweetened beverages. Fibroblast growth factor 21 (FGF21) is a complex metabolic regulator, and there is significant evidence that it may play a role in fructose metabolism, driving relative aversion to sweet taste. As such, we examined the relationship between FGF21 and the preferential intake of simple carbohydrates in mice, both as liquid solutions and as dietary additives. Genetic deletion of FGF21 or its obligate co-receptor β-klotho (KLB) had no impact on preference for sugar sweetened solutions. FGF21 overexpression, however, substantially suppressed preference for fructose solutions, but had no effect on glucose or sucrose preference. Infusions of FGF21 also suppressed fructose preference specifically, an effect that was dependent on expression of KLB in the CNS. These results demonstrate that FGF21 creates sugar-specific taste aversion to fructose, which may be mediated by a KLB-dependent pathway in the brain.HighlightsFGF21 administration suppresses fructose preference in mice.Preference for glucose or sucrose is not affected by FGF21 administration.Genetic FGF21 deletion does not enhance fructose, glucose, or sucrose preference.FGF21 requires central β-klotho expression to suppress fructose preference.

BACKGROUND: Fibroblast Growth Factor 21 (FGF21) is expressed in several metabolically active tissues, including liver, fat and acinar pancreas, and has pleiotropic effects on metabolic homeostasis. The dominant source of FGF21 in the circulation is the liver. OBJECTIVE AND METHODS: To analyze the physiological functions of hepatic FGF21, we generated a hepatocyte specific knockout model (LKO) by mating albumin-Cre mice with FGF21 flox/flox (fl/fl) mice and challenged it with different nutritional models. RESULTS: Mice fed a ketogenic diet typically show increased energy expenditure; this effect was attenuated in LKO mice. LKO on KD also developed hepatic pathology and altered hepatic lipid homeostasis. When evaluated using hyperinsulinemic euglycemic clamps, glucose infusion rates, hepatic glucose production and glucose uptake were similar between fl/fl and LKO DIO mice. CONCLUSIONS: We conclude that liver derived FGF21 is important for complete adaptation to ketosis but has a more limited role in the regulation of glycemic homeostasis.

Background Ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), also known as CD39L3, is the dominant ectonucleotidase expressed by beta cells in the islet of Langerhans and on nerves. NTPDase3 catalyzes the conversion of extracellular ATP and ADP to AMP and modulates purinergic signaling. Previous studies have shown that NTPDase3 decreases insulin release from beta-cells in vitro. This study aims to determine the impact of NTPDase3 in diet-induced obesity (DIO) and metabolism in vivo. Methods We developed global NTPDase3 deficient (Entpd3−/−) and islet beta-cell-specific NTPDase-3 deficient mice (Entpd3flox/flox,InsCre) using Ins1-Cre targeted gene editing to compare metabolic phenotypes with wildtype (WT) mice on a high-fat diet (HFD). Results Entpd3−/− mice exhibited similar growth rates compared to WT on chow diet. When fed HFD, Entpd3−/− mice demonstrated significant resistance to DIO. Entpd3−/− mice consumed more calories daily and exhibited less fecal calorie loss. Although Entpd3−/− mice had no increases in locomotor activity, the mice exhibited a significant increase in basal metabolic rate when on the HFD. This beneficial phenotype was associated with improved glucose tolerance, but not higher insulin secretion. In fact, Entpd3flox/flox,InsCre mice demonstrated similar metabolic phenotypes and insulin secretion compared to matched controls, suggesting that the expression of NTPDase3 in beta-cells was not the primary protective factor. Instead, we observed a higher expression of uncoupling protein 1 (UCP-1) in brown adipose tissue and an augmented browning in inguinal white adipose tissue with upregulation of UCP-1 and related genes involved in thermogenesis in Entpd3−/− mice. Conclusions Global NTPDase3 deletion in mice is associated with resistance to DIO and obesity-associated glucose intolerance. This outcome is not driven by the expression of NTPDase3 in pancreatic beta-cells, but rather likely mediated through metabolic changes in adipocytes.

Objective Reproduction is an energetically expensive process. Insufficient calorie reserves, signaled to the brain through peripheral signals such as leptin, suppress fertility. Recently, fibroblast growth factor 21 (FGF21) was implicated as a signal from the liver to the hypothalamus that directly inhibits the hypothalamic–gonadotropin axis during fasting and starvation. However, FGF21 itself increases metabolic rate and can induce weight loss, which suggests that the effects of FGF21 on fertility may not be direct and may reflect changes in energy balance. Methods To address this important question, we evaluated fertility in several mouse models with elevated FGF21 levels including ketogenic diet fed mice, fasted mice, mice treated with exogenous FGF21 and transgenic mice over-expressing FGF21. Results We find that ketogenic diet fed mice remain fertile despite significant elevation in serum FGF21 levels. Absence of FGF21 does not alter transient infertility induced by fasting. Centrally infused FGF21 does not suppress fertility despite its efficacy in inducing browning of inguinal white adipose tissue. Furthermore, a high fat diet (HFD) can restore fertility of female FGF21-overexpressing mice, a model of growth restriction, even in the presence of supraphysiological serum FGF21 levels. Conclusions We conclude that FGF21 is not a direct physiological regulator of fertility in mice. The infertility observed in FGF21 overexpressing mice is likely driven by the increased energy expenditure and consequent excess calorie requirements resulting from high FGF21 levels., Highlights • Ketogenic diet fed mice remain fertile despite significant elevation in serum FGF21. • Central infusion of FGF21 does not suppress fertility in female mice. • Mice lacking FGF21 have similar post-fasting delay of cycling as control mice. • High fat diet restores fertility in FGF21-Tg mice despite supra physiological serum FGF21.

Security is an essential part of development in embedded systems. Execution of unknown or malicious program through an unauthorized means of communication on an embedded system can cause unwanted system behavior. To safeguard the sensitive data and devices, presently, sophisticated hardware and software systems based on cryptographic techniques are required which in turn increases the system’s cost. In this paper, we proposed a method of securing such embedded devices which cannot afford to have capabilities comparable to conventional computers. This method generates a run-time trace on embedded devices during program execution, using already available hardware circuitry on the board. It observes and analyzes the obtained data using data analysis techniques and detects whether any change is occurred in the program compared to previously obtained data.

Pharmacologic inhibition of the renal sodium/glucose cotransporter-2 induces glycosuria and reduces glycemia. Given that SGLT2 inhibitors (SGLT2i) reduce mortality and cardiovascular risk in type 2 diabetes, improved understanding of molecular mechanisms mediating these metabolic effects is required. Treatment of obese but nondiabetic mice with the SGLT2i canagliflozin (CANA) reduces adiposity, improves glucose tolerance despite reduced plasma insulin, increases plasma ketones, and improves plasma lipid profiles. Utilizing an integrated transcriptomic-metabolomics approach, we demonstrate that CANA modulates key nutrient-sensing pathways, with activation of 5′ AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR), independent of insulin or glucagon sensitivity or signaling. Moreover, CANA induces transcriptional reprogramming to activate catabolic pathways, increase fatty acid oxidation, reduce hepatic steatosis and diacylglycerol content, and increase hepatic and plasma levels of FGF21. Given that these phenotypes mirror the effects of FGF21 to promote lipid oxidation, ketogenesis, and reduction in adiposity, we hypothesized that FGF21 is required for CANA action. Using FGF21-null mice, we demonstrate that FGF21 is not required for SGLT2i-mediated induction of lipid oxidation and ketogenesis but is required for reduction in fat mass and activation of lipolysis. Taken together, these data demonstrate that SGLT2 inhibition triggers a fasting-like transcriptional and metabolic paradigm but requires FGF21 for reduction in adiposity.

Objective Excess ethanol consumption has serious pathologic consequences. In humans, repeated episodes of binge drinking can lead to liver damage and have adverse effects on other organs such as pancreas and brain. Long term chronic consumption of ethanol can also result in progressive alcoholic liver disease and cirrhosis. Fibroblast growth factor 21 (FGF21) is a metabolic regulator with multiple physiologic functions. FGF21 is a novel biomarker for non-alcoholic fatty liver disease (NAFLD) in humans and limits hepatotoxicity in mice. Therefore, we explored the possibility that FGF21 plays a role in response to ethanol consumption in both humans and mice. Methods We used a binge drinking paradigm in humans to examine the effect of acute ethanol consumption on circulating FGF21. We adapted this paradigm to evaluate the acute response to ethanol in mice. We then examined the role of FGF21 on liver pathology in two models of chronic ethanol consumption in both wild type (WT) mice and mice lacking FGF21 (FGF21-KO). Results Acute ethanol consumption resulted in a robust induction of serum FGF21 after 6 h in both humans and mice. Serum ethanol peaked at 1 h in both species and was cleared by 6 h. Ethanol clearance was the same in WT and FGF21-KO mice, indicating that FGF21 does not play a major role in ethanol metabolism in a binge paradigm. When FGF21-KO mice were fed the Lieber–DeCarli diet, a high fat diet supplemented with ethanol, a higher mortality was observed compared to WT mice after 16 days on the diet. When FGF21-KO mice consumed 30% ethanol in drinking water, along with a normal chow diet, there was no mortality observed even after 16 weeks, but the FGF21-KO mice had significant liver pathology compared to WT mice. Conclusions Acute or binge ethanol consumption significantly increases circulating FGF21 levels in both humans and mice. However, FGF21 does not play a role in acute ethanol clearance. In contrast, chronic ethanol consumption in the absence of FGF21 is associated with significant liver pathology alone or in combination with excess mortality, depending on the type of diet consumed with ethanol. This suggests that FGF21 protects against long term ethanol induced hepatic damage and may attenuate progression of alcoholic liver disease. Further study is required to assess the therapeutic potential of FGF21 in the treatment of alcoholic liver disease., Highlights • Binge ethanol consumption robustly increases circulating FGF21 levels in both humans and mice. • FGF21 does not play a role in ethanol clearance which is the same in WT and FGF21-KO mice. • In mice lacking FGF21, in two models of ethanol consumption there was either excess mortality or excess hepatic damage and inflammation. • These data suggest the FGF21 may have potential therapeutic value for alcoholic liver disease.

Mesenchymal epithelial transition factor (MET) is one of the critical cell signaling molecules whose aberrant expression is reported in several human cancers. The aim of the study is to investigate the antigene and antiproliferative effect of short triplex forming oligonucleotides, TFO-1 (part of the positive regulatory element) and TFO-2 (away from the transcription start site) on MET expression. HepG2 cells transfected only with TFO-1 (but not with TFO-2 and non-specific TFO) significantly decreased MET levels, which is accompanied by decrease in antiapoptotic proteins and increase in pro-apoptotic proteins. Phosphoproteome-array analysis of 46 intracellular kinases revealed hypophosphorylation of about 15 kinases including ERK, AKT, Src and MEK, suggesting the growth inhibitory effect of TFO-1. Further, the efficacy of TFO-1 was tested on diethylnitrosamine-induced liver tumors in wistar rats. T2-weighted magnetic resonance imaging showed decrease in liver tumor volume up to 90% after treatment with TFO-1. Decreased MET expression and elevated apoptotic activity further indicate that TFO-1 targeted to c-met leads to cell death and tumor regression in hepatoma. Formation of stable DNA triplex between TFO-1 and targeted gene sequence was confirmed by circular dichroic spectroscopy and gel retardation assay. Therefore, it can be concluded that DNA triplex-based therapeutic approaches hold promise in the treatment of malignancies associated with MET overexpression.

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma. Mol Cancer Res; 9(6); 801–12. ©2011 AACR.

Development of green nanotechnology is generating interest of researchers toward ecofriendly biosynthesis of nanoparticles. In this study, biosynthesis of stable silver nanoparticles was done using Tulsi (Ocimum sanctum) leaf extract. These biosynthesized nanoparticles were characterized with the help of UV–vis spectrophotometer, Atomic Absorption Spectroscopy (AAS), Dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). Stability of bioreduced silver nanoparticles was analyzed using UV–vis absorption spectra, and their antimicrobial activity was screened against both gram-negative and gram-positive microorganisms. It was observed that O. sanctum leaf extract can reduce silver ions into silver nanoparticles within 8 min of reaction time. Thus, this method can be used for rapid and ecofriendly biosynthesis of stable silver nanoparticles of size range 4–30 nm possessing antimicrobial activity suggesting their possible application in medical industry.

c-Met, the tyrosine kinase receptor for hepatocyte growth factor plays a pivotal role in normal cellular signaling and overexpression of c-Met protein is reported in several human cancers. Thus, transcriptional regulation of c-met appears to be an attractive target for chemotherapy. Therefore, we selected a 24mer GC rich sequence (24R) from the c-met promoter located at -142 to -119 from transcription start site and studied its interaction with anticancer drug, Actinomycin D. Spectroscopic analysis demonstrated a strong complexation between ActD and 24RY as shown by: (i) a high binding constant, K of 4-5 x 10(5) M(-1) with DeltaDeltaG of -47 +/- 1.5 Kcalmol(-1); (ii) marked increase by +10 degrees C in melting temperature of 24RY; and (iii) significant changes in circular dichroic spectra of both ActD and 24RY. Molecular modeling revealed the preference of ActD to the Sp1 binding site, GGCGGG, in 24RY. Expression of the c-Met was checked in HepG2 cells, a human hepatocellular carcinoma cell line by using western blotting and immunocytochemistry. Downregulation of c-Met expression by as much as 50% was observed in the presence of 20ng/ml (IC(50)) of ActD. Taking into account of the binding studies also, we feel that the down regulation of c-Met perhaps involves binding of ActD to the promoter site of c-met. Therefore, c-met could be a challenging and promising target for therapeutic strategies in combating cancer.

Background The metabolic regulator Fibroblast Growth Factor 21 (FGF21) is highly expressed in the acinar pancreas, but its role in pancreatic function is obscure. It appears to play a protective role in acute experimental pancreatitis in mice. The aim of this study was to define an association between FGF21 and the course and resolution of acute pancreatitis in humans. Methods and Principal Findings Twenty five subjects with acute pancreatitis admitted from May to September 2012 to the Beth Israel Deaconess Medical Center (BIDMC) were analyzed. Serial serum samples were collected throughout hospitalization and analyzed for FGF21 levels by ELISA. Twenty healthy subjects sampled three times over a four week period were used as controls. We found that, in patients with pancreatitis, serum FGF21 rises significantly and peaks four to six days after the maximum lipase level, before slowly declining. Maximum FGF21 levels were significantly greater than baseline levels for acute pancreatitis subjects (1733 vs. 638 pg/mL, P = 0.003). This maximum value was significantly greater than the highest value observed for our control subjects (1733 vs. 322 pg/mL, P = 0.0002). The ratio of active to total FGF21 did not change during the course of the disease (42.5% vs. 44.4%, P = 0.58). Fold increases in FGF21 were significantly greater in acute pancreatitis subjects than the fold difference seen in healthy subjects (4.7 vs. 2.0, P = 0.01). Higher fold changes were also seen in severe compared to mild pancreatitis (18.2 vs. 4.4, P = 0.01). The timing of maximum FGF21 levels correlated with day of successful return to oral intake (R2 = 0.21, P = 0.04). Conclusions Our results demonstrate that serum FGF21 rises significantly in humans with acute pancreatitis. The pancreas may be contributing to increased FGF21 levels following injury and FGF21 may play a role in the recovery process.

The nicotinic acetylcholine receptor (AChR) is a long-term, surface resident molecule, and its localization and maintenance at synapses is key for communication as well as during development. AChR is one of the best characterized ligand gated ion channels, and its distribution in the plasma membrane is important for synaptic plasticity. Using STED microscopy the AChR has been shown to be present in ∼80 nm structures cell surface structures which increase in size upon ligation (Kellner et al., 2007). Furthermore, binding of the competitive antagonist α-bungarotoxin, or a monoclonal antibody against its α-subunit, induces a clathrin and dynamin independent internalization via surface-connected tubular compartments, eventually reaching lysosomes for degradation (Kumari et al., 2008). These observations suggest a functional coupling between scales of organization and the endocytic machinery. Here, using homo-FRET and TIRF microscopy we explore the nanometric and optically resolved scale of AChR organization at the surface of the living cell in its native state, as well as after various perturbations of membrane and cortical actin composition and structure. These studies make apparent the relationship between AChR internalization and actin dynamics and throw light on the possible mechanisms involved in the stability of the AChR at the postsynaptic membrane.

The c-Met signaling pathway is one of the main regulators of cell proliferation, differentiation, apoptosis, and other cellular processes. As aberrant expression of c-Met has been implicated in tumor growth, invasion, and metastasis, c-Met is recognized as a promising target for cancer therapeutics. In continuation of our search for small molecules with anticancer properties, we investigated the binding of bis-benzimidazole derivative Hoechst-33258 to the promoter region 24RY, -142 to -119 (5'-GGGGCAGAGGCGGGAGGAAACGCG-3', 24R and its complement 5'-CGCGTTTCCTCCCGCCTCTGCCCC-3', 24Y), upstream to the transcriptional start site of the c-met gene. Strong complexation of duplex 24RY-Hoechst is revealed by a high binding constant K, 3.25 x 10(5) M(-1), with significant changes of Delta DeltaG (-10 kcal/mol), Delta DeltaH (-83 kcal/mol), Delta DeltaS (-241 cal/[mol K]), and melting temperature (DeltaT(m) = +8 degrees C). This is accompanied by bathochromic shift of 10 nm, 50% hypochromism at 340 nm, isobestic points at 302 and 369 nm in absorption spectra, and induced emission band at 465 nm in the fluorescence spectra of Hoechst. Molecular modeling data demonstrated that Hoechst binds to consecutive GGs in the minor groove of 24RY containing residues 12-17. Therefore, Hoechst-33258 can be a potential anticancer agent against malignancies associated with c-Met upregulation.