Skin Cancer Detection in Diverse Skin Tones by Machine Learning Combining Audio and Visual Convolutional Neural Networks.

INTRODUCTION: Skin cancer (SC) is common in fair skin (FS) at a 1:5 lifetime incidence for nonmelanoma skin cancer. In order to assist clinicians' decisions, a risk intervention technology was developed, which combines a dual-mode machine learning of visual and sonified (pixel to sound) data. The addition of an audio technology enhances malignant features of lesions, increases sensitivity and was previously validated under a prospective clinical setting in FS. In dark skin (DS), although rare by a 10-30 factor, skin cancer is diagnosed at more advanced stages resulting in a delayed diagnosis and affecting life quality and expectancy. It is known as well that SC diagnostic accuracy by machine learning in DS is decreased as compared to FS. The present study tests the use of sonification aided by artificial intelligence algorithms to compare diagnostics of different skin tones. METHODOLOGY: Biopsy-validated smartphone images were diagnosed in a retrospective study by a dual audio-visual convoluted neural network. A total of 60 Fitzpatrick I-III were compared to 72 Fitzpatrick IV-VI. A dichotomous diagnostic output, either malignant or benign, was assessed for sensitivity, specificity and area under the curves (AUCs) for the receiver operating characteristic (ROC) curve. RESULTS: ROC curve analytics indicated an AUC of 0.858 (95% CI: 0.795-0.921) and 0.856 (95% CI: 0.759-0.953) for fair and DS (p = NS). Sensitivity of Fitzpatrick I-III skin and Fitzpatrick IV-VI were 84.4% (71.8-96.9) and 79.6% (63.4-93.8), respectively (p = NS). Specificity of Fitzpatrick I-III skin and Fitzpatrick IV-VI were 84.2% (72.6-95.8) and 85.3% (73.4-97.2), respectively (p = NS). The positive predictive and negative predictive values as well as accuracy (0.817 vs. 0.847) were all within the same range (p = NS). CONCLUSIONS: The results demonstrate that the dual-modality classifier identifies skin cancer of FS and DS similarly well. Sonification of malignant signs of a skin lesion demonstrates promising results, even with smartphone images, which should be considered as a tool to achieve more effective and accessible healthcare.

Tracking Ca2+-dependent and Ca2+-independent conformational transitions in syntaxin 1A during exocytosis in neuroendocrine cells.

A key issue for understanding exocytosis is elucidating the various protein interactions and the associated conformational transitions underlying soluble N-ethylmeleimide-sensitive factor attachment protein receptor (SNARE) protein assembly. To monitor dynamic changes in syntaxin 1A (Syx) conformation along exocytosis, we constructed a novel fluorescent Syx-based probe that can be efficiently incorporated within endogenous SNARE complexes, support exocytosis, and report shifts in Syx between 'closed' and 'open' conformations by fluorescence resonance energy transfer analysis. Using this probe we resolve two distinct Syx conformational transitions during membrane depolarization-induced exocytosis in PC12 cells: a partial 'opening' in the absence of Ca(2+) entry and an additional 'opening' upon Ca(2+) entry. The Ca(2+)-dependent transition is abolished upon neutralization of the basic charges in the juxtamembrane regions of Syx, which also impairs exocytosis. These novel findings provide evidence of two conformational transitions in Syx during exocytosis, which have not been reported before: one transition directly induced by depolarization and an additional transition that involves the juxtamembrane region of Syx. The superior sensitivity of our probe also enabled detection of subtle Syx conformational changes upon interaction with VAMP2, which were absolutely dependent on the basic charges of the juxtamembrane region. Hence, our results further suggest that the Ca(2+)-dependent transition in Syx involves zippering between the membrane-proximal juxtamembrane regions of Syx and VAMP2 and support the recently implied existence of this zippering in the final phase of SNARE assembly to catalyze exocytosis.

Platelet-derived growth factor BB mimics serum-induced dispersal of pancreatic epithelial cell clusters.

We showed previously that proliferating human islet-derived de-differentiated cells (DIDs) exhibit many characteristics of mesenchymal stem cells. Dispersed DIDs can be induced by serum deprivation to undergo mesenchymal-to-epithelial transition and aggregate into epithelial cell clusters (ECCs). Conversely, ECCs can be induced to disperse and undergo epithelial-to-mesenchymal transition (EMT) by re-addition of mammalian sera. In this study, we show that platelet-derived growth factor BB (PDGF-BB) mimics and mediates serum-induced ECCs' dispersal accompanied by accumulation of cytoplasmic ß-catenin and a decrease in the levels of insulin and glucagon mRNAs. Moreover, we show that PDGF-BB-induced dispersal of ECCs is a more general phenomenon that occurs also with bone marrow mesenchymal stem cells (BM-MSCs) and dermal fibroblasts (DFs). In DIDs, BM-MSCs, and DFs, PDGF decreased the levels of DKK1 mRNA, suggesting involvement of the Wnt signaling pathway. PDGF-BB stimulated a significant increase in S473 phosphorylation of Akt and the PI3K specific inhibitor (PIP828) partially inhibited PDGF-BB-induced ECC dispersal. Lastly, the PDGF-receptor (PDGF-R) antagonist JNJ-10198409 inhibited both PDGF-BB--and serum-induced ECC dispersal. Epidermal growth factor (EGF), which shares most of the PDGF signaling pathway, did not induce dispersal and only weakly stimulated Akt phosphorylation. Our data suggest that PDGF-BB mediates serum-induced DIDs dispersal, correlated with the activation of the PI3K-Akt pathway.

INDI: a computational framework for inferring drug interactions and their associated recommendations.

Inferring drug-drug interactions (DDIs) is an essential step in drug development and drug administration. Most computational inference methods focus on modeling drug pharmacokinetics, aiming at interactions that result from a common metabolizing enzyme (CYP). Here, we introduce a novel prediction method, INDI (INferring Drug Interactions), allowing the inference of both pharmacokinetic, CYP-related DDIs (along with their associated CYPs) and pharmacodynamic, non-CYP associated ones. On cross validation, it obtains high specificity and sensitivity levels (AUC (area under the receiver-operating characteristic curve) ≥0.93). In application to the FDA adverse event reporting system, 53% of the drug events could potentially be connected to known (41%) or predicted (12%) DDIs. Additionally, INDI predicts the severity level of each DDI upon co-administration of the involved drugs, suggesting that severe interactions are abundant in the clinical practice. Examining regularly taken medications by hospitalized patients, 18% of the patients receive known or predicted severely interacting drugs and are hospitalized more frequently. Access to INDI and its predictions is provided via a web tool at http://www.cs.tau.ac.il/~bnet/software/INDI, facilitating the inference and exploration of drug interactions and providing important leads for physicians and pharmaceutical companies alike.

Regional Antibiotic Delivery for Implanted Cardiovascular Electronic Device Infections.

BACKGROUND: Present guidelines endorse complete removal of cardiovascular implantable electronic devices (pacemakers/defibrillators), including extraction of all intracardiac electrodes, not only for systemic infections, but also for localized pocket infections. OBJECTIVES: The authors evaluated the efficacy of delivering continuous, in situ-targeted, ultrahigh concentration of antibiotics (CITA) into the infected subcutaneous device pocket, obviating the need for device/lead extraction. METHODS: The CITA group consisted of 80 patients with pocket infection who were treated with CITA during 2007-2021. Of them, 9 patients declined lead extraction because of prohibitive operative risk, and 6 patients had questionable indications for extraction. The remaining 65 patients with pocket infection, who were eligible for extraction, but opted for CITA treatment, were compared with 81 patients with pocket infection and similar characteristics who underwent device/lead extraction as primary therapy. RESULTS: A total of 80 patients with pocket infection were treated with CITA during 2007-2021. CITA was curative in 85% (n = 68 of 80) of patients, who remained free of infection (median follow-up 3 years [IQR: 1.0-6.8 years]). In the case-control study of CITA vs device/lead extraction, cure rates were higher after device/lead extraction than after CITA (96.2% [n = 78 of 81] vs 84.6% [n = 55 of 65]; P = 0.027). However, rates of serious complications were also higher after extraction (n = 12 [14.8%] vs n = 1 [1.5%]; P = 0.005). All-cause 1-month and 1-year mortality were similar for CITA and device/lead extraction (0.0% vs 3.7%; P = 0.25 and 12.3% vs 13.6%; P = 1.00, respectively). Extraction was avoided in 90.8% (n = 59 of 65) of extraction-eligible patients treated with CITA. CONCLUSIONS: CITA is a safe and effective alternative for patients with pocket infection who are unsuitable or unwilling to undergo extraction. (Salvage of Infected Cardiovascular Implantable Electronic Devices [CIED] by Localized High-Dose Antibiotics; NCT01770067).

Knock-down of plasminogen-activator inhibitor-1 enhances expression of E-cadherin and promotes epithelial differentiation of human pancreatic adenocarcinoma cells.

High levels of plasminogen activator inhibitor-1 (PAI-1), which is produced by stromal, endothelial, and cancer cells and has multiple complex effects on cancers, correlate with poor cancer prognosis. To more definitively study the role of endogenously produced PAI-1 in human pancreatic adenocarcinoma (PAC) PANC-1 cell line biology, we used anti-PAI-1 shRNA to create stable PAI-1 deficient cells (PD-PANC-1s). PD-PANC-1s exhibited a heterogeneous morphology. While the majority of cells exhibited a cuboidal shape similar to the parental PANC-1 or the vector-infected control cells, numerous large cells with long filopodia and a neuronal-like appearance were observed. Although both Vector-control cells and PD-PANC-1s expressed mRNAs that are characteristic of mesenchymal, neural, and epithelial phenotypes, epithelial marker RNAs were up-regulated (e.g., E-cadherin, 32-fold) whereas mesenchymal marker RNAs were down-regulated (e.g., Thy1, ninefold) in PD-PANC-1s, suggesting mesenchymal-to-epithelial transition. Neural markers exhibited both up- and down-regulation. Immunocytochemistry indicated that epithelial-like PD-PANC-1s expressed E-cadherin and ß-catenin in significantly more cells, while neural-like cells exhibited robust expression of organized ß-3-tubulin. PAI-1 and E-cadherin were rarely co-expressed in the same cells. Indeed, examination of PAI-1 and E-cadherin mRNAs expression in additional cell lines yielded clear inverse correlation. Indeed, infection of Colo357 PAC cells (that exhibit high expression of E-cadherin) with PAI-1-expressing adenovirus led to a marked decrease in E-cadherin expression and to enhanced migration of cells from clusters. Our results suggest that endogenous PAI-1 suppresses expression of E-cadherin and differentiation in PAC cells in vitro, supporting its negative impact on tumor prognosis.

MMP expression in leaking filtering blebs and tears after glaucoma filtering surgery.

OBJECTIVE: Glaucoma filtering surgery may be compromised by cystic blebs which develop more frequently when anti-metabolites are used to arrest wound healing. Matrix metalloproteinases (MMPs) and the naturally occurring tissue inhibitors of metalloproteinases (TIMPs) are essential in connective tissue remodeling and wound healing. This study aimed to determine whether filtering blebs display increased expression of MMP-2, MMP-9, TIMP-1 and TIMP-2, and whether it is reflected in tear fluid. METHODS: Tissue samples from leaking blebs (n = 5) and control conjunctiva (n = 5) were evaluated by immunohistochemistry for MMP-2, MMP-9, TIMP-1 and TIMP-2. Tear fluid was collected from 12 patients (12 eyes) with cystic blebs and ten patients (ten eyes) with flat blebs following trabeculectomy with Mitomycin C applied and 16 controls. MMP levels were evaluated by zymography and TIMP levels by Western blot analysis. RESULTS: Conjunctival tissue was obtained from five eyes with cystic leaking blebs and five control eyes undergoing cataract surgery. More extensive MMP-2 and MMP-9 expression was found in the epithelial and stromal layers of blebs than in control conjunctiva. TIMP-1and TIMP-2 were expressed in all layers of the blebs, but only in the epithelium of control conjunctiva. MMP-2 and proMMP-2 activity in tears from eyes with flat blebs was significantly higher than that of controls, while activity in tears of eyes with cystic blebs was significantly higher than in those with flat blebs. There was no difference in MMP-9 activity between tears of control and post-filtering surgery eyes. CONCLUSIONS: Increased MMPs and TIMPs expression is associated with the formation of filtering blebs, suggesting involvement of MMPs in bleb remodeling. MMP-2 and ProMMP-2 levels in tear fluid may be markers for bleb configuration.

Human islet-derived precursor cells can cycle between epithelial clusters and mesenchymal phenotypes.

We showed previously that undifferentiated, proliferating human islet-derived precursor cells (hIPCs) are a type of mesenchymal stem/stromal cell (MSC) that can be induced by serum deprivation to form clusters and ultimately differentiate in vitro to endocrine cells. We also demonstrated that partially differentiated hIPC clusters, when implanted under the kidney capsules of mice, continued to differentiate in vivo into hormone-producing cells. However, we noted that not all hIPC preparations yielded insulin-secreting cells in vivo and that in some animals no hormone-expressing cells were found. This suggested that the implanted cells were not always irreversibly committed to further differentiation and may even de-differentiate to a mesenchymal phenotype. In this study, we show that human cells with a mesenchymal phenotype are indeed found in the grafts of mice implanted with hIPCs in epithelial cell clusters (ECCs), which are obtained after 4-day in vitro culture of hIPCs in serum-free medium (SFM); mesenchymal cells were predominant in some grafts. We could mimic the transition of ECCs to de-differentiated mesenchymal cells in vitro by exposure to foetal bovine serum (FBS) or mouse serums, and to a significantly lesser extent to human serum. In a complementary series of experiments, we show that mouse serum and FBS are more effective stimulants of mesenchymal hIPC migration than is human serum. We found that proliferation was not needed for the transition from ECCs to de-differentiated cells because mitomycin-treated hIPCs that could not proliferate underwent a similar transition. Lastly, we show that cells exhibiting a mesenchymal phenotype can be found in grafts of adult human islets in mice. We conclude that epithelial-to-mesenchymal transition (EMT) of cells in hIPC ECCs can occur following implantation in mice. This potential for EMT of human islets or differentiated precursor cells must be considered in strategies for cell replacement therapy for diabetes.

Kaposi's sarcoma-associated herpesvirus-G protein-coupled receptor-expressing endothelial cells exhibit reduced migration and stimulated chemotaxis by chemokine inverse agonists.

A constitutively active G protein-coupled receptor (GPCR) encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) (KSHV) is expressed in endothelial (spindle) cells of Kaposi's sarcoma lesions. In this study, we report novel effects of basal signaling by this receptor and of inverse agonist chemokines on migration of KSHV-GPCR-expressing mouse lung endothelial cells. We show that basal signaling by KSHV-GPCR inhibits migration of endothelial cells in two systems, movement through porous filters and in vitro wound closure. Naturally occurring chemokines, interferon gamma-inducible protein-10 and stromal-derived factor-1, which act as inverse agonists at KSHV-GPCR, abrogate the inhibition of migration and stimulate directed migration (or chemotaxis) of these cells. Thus, the expression of KSHV-GPCR may allow infected endothelial cells in situ to remain in a localized environment or to directionally migrate along a gradient of specific chemokines that are inverse agonists at KSHV-GPCR.

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

G protein-activated K+ channels: a reporter for rapid activation of G proteins by lysophosphatidic acid in Xenopus oocytes.

Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)-sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein-activated K+ channel (GIRK) as a reporter. Low [LPA] induced IK+ in <3 s of the agonist addition with little or no activation of chloride current. Depletion of Galphao/Galphao1 each decreased the LPA-induced IK+ by approximately 40-50%, while PTX completely abolished it. This is the first direct evidence showing the activation of GIRK by LPA, and the involvement of G proteins of the Go family in rapid desensitization of LPA responses.

COX-2 inhibition improves retinal function in rats' ischemic eyes.

PURPOSE: Retinal ischemia is a relatively simple model for studies in pharmacological neuroprotective intervention. The role of cyclooxygenase (COX) enzymes in ischemic insult has been variously shown to either increase or decrease ischemic damage. The purpose of this study was to assess the role of COX-1 and COX-2 in rat retinal ischemic functional damage. METHODS: Ischemia was achieved by elevating intraocular pressure for 60 min. White flash electroretinogram (ERG) was recorded by contact lens electrodes containing an integral light emitting diode source. ERG was recorded on post-ischemia (PI) days-1 (baseline), 1, 3, and 7. The b-wave amplitude, b-wave implicit time, and oscillatory potentials (OPs) were analyzed. The expression of COX-2 and HSP70i was assessed by Western analysis on day 1 PI. RESULTS: Ischemia caused attenuation of OPs, a decrease in the b-wave amplitude, and an increase in b-wave implicit time, accompanied by the increased expression of COX-2 and HSP70i proteins. Selective COX-2 inhibition markedly increased b-wave amplitude and enhanced retinal HSP70i induction, whereas COX-1 or nonselective and irreversible inhibition of both COX isoenzymes did not affect the retinal function or the expression of these proteins. High-dose aspirin prevented partial recovery from ischemic damage. Administration of a synthetic PGF2α analog, or a lipoxygenase inhibitor, had little effect on ischemic damage, but affected nonischemic ERG. CONCLUSIONS: COX-2 appears to mediate some of the ischemic retinal functional damage, possibly by inhibiting the induction of HSP-70i. We propose that selective COX-2 inhibitors may be useful in pathological conditions involving ischemic retinal insult.

Proteinase-activated receptors differentially modulate in vitro invasion of human pancreatic adenocarcinoma PANC-1 cells in correlation with changes in the expression of CDC42 protein.

OBJECTIVES: Proteinase-activated receptor-1 (PAR-1) and PAR-2 have been associated with increased invasiveness and metastasis in human malignancies. The role of PAR-3 has been less investigated. We examined the role of PARs in a human pancreatic adenocarcinoma PANC-1 cell line phenotype in vitro. METHODS: We knocked down PAR-1, PAR-2, or PAR-3, whereas empty vector-infected cells served as controls. Specific peptide agonists of PARs were used to stimulate the receptors. In vitro assays of colony formation, migration, and invasion were used to characterize the phenotypes, and Western analysis was used to follow cell division control protein 42 homolog (CDC42) expression. RESULTS: PAR-1 and PAR-2 knockdowns (KDs) were markedly less, whereas PAR-3 KDs were robustly more migratory and invasive than the controls. Stimulation of PAR-1 or PAR-2 by their peptide agonists increased, whereas PAR-3 agonist reduced the invasion of the control cells. Knockdowns of all three PARs exhibited changes in the expression of CDC42, which correlated with the changes in their invasion. Conversely, stimulation of vector-control cells with PAR-1 or PAR-2 agonists enhanced, whereas PAR-3 agonist reduced the expression of CDC42. In the respective KD cells, the effects of the agonists were abrogated. CONCLUSION: The expression and/or activation of PARs is linked to the invasiveness of PANC-1 cells in vitro, probably via modulation of the expression of CDC42.

PAR-3 knockdown enhances adhesion rate of PANC-1 cells via increased expression of integrinαv and E-cadherin.

The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.

Serum deprivation induces glucose response and intercellular coupling in human pancreatic adenocarcinoma PANC-1 cells.

OBJECTIVE: This study aimed to investigate whether the previously described differentiating islet-like aggregates of human pancreatic adenocarcinoma cells (PANC-1) develop glucose response and exhibit intercellular communication. METHODS: Fura 2-loaded PANC-1 cells in serum-free medium were assayed for changes in cytosolic free calcium ([Ca]i) induced by depolarization, tolbutamide inhibition of K(ATP) channels, or glucose. Dye transfer, assayed by confocal microscopy or by FACS, was used to detect intercellular communication. Changes in messenger RNA (mRNA) expression of genes of interest were assessed by quantitative real-time polymerase chain reaction. Proliferation was assayed by the MTT method. RESULTS: Serum-deprived PANC-1 cell aggregates developed [Ca]i response to KCl, tolbutamide, or glucose. These responses were accompanied by 5-fold increase in glucokinase mRNA level and, to a lesser extent, of mRNAs for K(ATP) and L-type calcium channels, as well as increase in mRNA levels of glucagon and somatostatin. Trypsin, a proteinase-activated receptor 2 agonist previously shown to enhance aggregation, modestly improved [Ca]i response to glucose. Glucose-induced coordinated [Ca]i oscillations and dye transfer demonstrated the emergence of intercellular communication. CONCLUSIONS: These findings suggest that PANC-1 cells, a pancreatic adenocarcinoma cell line, can be induced to express a differentiated phenotype in which cells exhibit response to glucose and form a functional syncytium similar to those observed in pancreatic islets.

Prophylactic treatment with telmisartan induces tissue-specific gene modulation favoring normal glucose homeostasis in Cohen-Rosenthal diabetic hypertensive rats.

The objectives were to assess the potential of long-term prophylactic administration of telmisartan, an angiotensin II receptor antagonist and a partial peroxisome proliferator activator receptor (PPAR)γ agonist, in preventing the development of hypertension and hyperglycemia and to demonstrate the alteration in gene expression associated with the development of hyperglycemia and insulin resistance in Cohen-Rosenthal diabetic hypertensive rat, a unique model of hypertension and type 2 diabetes mellitus comorbidity. Cohen-Rosenthal diabetic hypertensive rats were continuously treated with telmisartan (3 mg/[kg d]) starting at age 6 to 8 weeks before developing hypertension or diabetes. Weight changes, blood pressure, blood insulin, adiponectin, glucose tolerance, and insulin sensitivity were monitored. Fat, liver, and muscle messenger RNAs were examined for the expression of genes potentially involved in the onset of insulin resistance. In addition to the expected antihypertensive effect of prophylactic telmisartan, diabetes was blunted, evidenced at the end of the study by a significantly lower glucose level. This was accompanied by improved glucose tolerance, increased sensitivity to insulin, reduction in fasting insulin levels and homeostasis model assessment index, as well as an increase in serum adiponectin. Telmisartan also prevented the increase in serum triglycerides and the associated appearance of lipid droplets in the liver. Diabetes induced tissue-specific changes in messenger RNAs expression of the following selected genes, which were restored by telmisartan treatment: PPARγ, PPARδ, PPARγ coactivator 1α, adiponectin, adiponectin receptor 1, adiponectin receptor 2, phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, adenosine monophosphate kinase, and glucose translocator 4. Telmisartan blunted the development of hypertension, insulin resistance, and diabetes in prediabetic Cohen-Rosenthal diabetic hypertensive rats through pleiotropic activity, involving specific gene regulation of target organs.

Telmisartan ameliorates hyperglycemia and metabolic profile in nonobese Cohen-Rosenthal diabetic hypertensive rats via peroxisome proliferator activator receptor-gamma activation.

The importance of hypertension treatment has expanded beyond blood pressure management to include additional risk factors, mainly diabetes. It was considered of interest to test the effect of telmisartan, an angiotensin receptor 1 antagonist and peroxisome proliferator activator receptor-gamma partial agonist, on Cohen-Rosenthal diabetic hypertensive nonobese (CRDH) rats, a unique model combining both pathologies. Its effect was examined on fat-derived and inflammatory agents in CRDH. To determine the extent of the drug's peroxisome proliferator activator receptor-gamma modulating beneficial metabolic actions, results were compared with those obtained with valsartan and rosiglitazone in CRDH and Cohen diabetic rat (CDR). Telmisartan and valsartan were given in drinking water at 3 and 12 mg/kg/d, whereas rosiglitazone (3 mg/kg/d) was given as food admixture for a period of 5 months. Blood pressure, glucose, insulin, adiponectin, leptin, and tumor necrosis factor alpha were examined. Telmisartan and valsartan significantly (P < .01) reduced blood pressure, whereas telmisartan and rosiglitazone considerably reduced blood glucose levels to normoglycemic levels (P < .01) in these 2 strains. Insulin levels were not affected by telmisartan and valsartan but were slightly reduced by rosiglitazone in CDR. In contrast to valsartan, adiponectin was significantly (60%, P < .01) increased by telmisartan in both CDR and CRDH, whereas rosiglitazone induced a 60% and 180% increase in CRDH and CDR animals, respectively, on day 30 of treatment. Co-treatment with GW9662 averted telmisartan-induced rise of adiponectin. Tumor necrosis factor alpha declined in telmisartan-treated rats, less so with rosiglitazone, but not valsartan. Telmisartan also induced downsizing of epididymal adipocytes compared with valsartan. Leptin levels were significantly increased by valsartan (P < .05) but reduced by telmisartan and rosiglitazone. The telmisartan-induced increase in adiponectin was most probably associated with a decrease in glucose and tumor necrosis factor alpha levels. Therefore, in addition to its hypotensive effect, telmisartan demonstrated beneficial thiazolidinedione-like effects.

A novel HMM-based method for detecting enriched transcription factor binding sites reveals RUNX3 as a potential target in pancreatic cancer biology.

BACKGROUND: Pancreatic adenocarcinoma (PAC) is one of the most intractable malignancies. In order to search for potential new therapeutic targets, we relied on computational methods aimed at identifying transcription factor binding sites (TFBSs) over-represented in the promoter regions of genes differentially expressed in PAC. Though many computational methods have been implemented to accomplish this, none has gained overall acceptance or produced proven novel targets in PAC. To this end we have developed DEMON, a novel method for motif detection. METHODOLOGY: DEMON relies on a hidden Markov model to score the appearance of sequence motifs, taking into account all potential sites in a promoter of potentially varying binding affinities. We demonstrate DEMON's accuracy on simulated and real data sets. Applying DEMON to PAC-related data sets identifies the RUNX family as highly enriched in PAC-related genes. Using a novel experimental paradigm to distinguish between normal and PAC cells, we find that RUNX3 mRNA (but not RUNX1 or RUNX2 mRNAs) exhibits time-dependent increases in normal but not in PAC cells. These increases are accompanied by changes in mRNA levels of putative RUNX gene targets. CONCLUSIONS: The integrated application of DEMON and a novel differentiation system led to the identification of a single family member, RUNX3, which together with four of its putative targets showed a robust response to a differentiation stimulus in healthy cells, whereas this regulatory mechanism was absent in PAC cells, emphasizing RUNX3 as a promising target for further studies.

The involvement of matrix metalloproteinases 2 and 9 in rat retinal ischemia.

BACKGROUND: The involvement of matrix metalloproteinases (MMPs) in ischemic tissue damage and remodeling has been reported by many investigators. Our study was designed to investigate the involvement of MMPs and of tissue inhibitors of metalloproteinases (TIMPs) in rat retinal ischemic injury, the effect of nitric oxide synthase (NOS) inhibitors on MMPs' activity in this model and whether minocycline (an MMP inhibitor) is protective in retinal ischemia. METHODS: Ninety-four rats were used in the study. Ischemia was induced by 90 min elevation of intraocular pressure. MMPs' activities and the effect of NOS inhibitors [aminoguanidine (AG) or N-nitro-L-arginine (NNA)] and minocycline on MMPs' activities were assessed by zymography and TIMPs expression by Western analysis. Morphological damage was quantified by morphometry of hematoxylin and eosin-stained retinal sections. RESULTS: Retinal extracts exhibited activities of proMMP-9 and proMMP-2. The activity of proMMP-9 increased immediately post ischemia (PI) and peaked to 4.6 times that of normal untreated controls in ischemic retinas and to 2.6 times that of controls in retinas of fellow sham-treated eyes at 24 h PI. The relative amount of TIMP-1 increased to 1.9-fold following ischemia and 2.5-fold in fellow sham-treated eyes at 24 h PI. ProMMP-2 activity increased more than two-fold immediately, at 24 h and at 48 h PI in ischemic retinas, and insignificantly in fellow sham-treated eyes. Treatment with 25 mg/kg AG or NNA caused a non-significant increase in proMMP-9 activity at 24 h PI (3.7- and 2.9-fold, respectively, p>0.6). There was no effect of AG or NNA on the activity of proMMP-2. Minocycline significantly attenuated the retinal ischemic damage, primarily by partially preserving ganglion cells and the inner plexiform layer. Minocyline (0.5 mg/ml or 5 mg/ml) inhibited MMPs' activities in ischemic retinal extracts in vitro. CONCLUSIONS: MMPs participated in morphological ischemic damage to rat retina. Treatment with minocycline dramatically attenuated damage to the retina.

Trypsin and thrombin accelerate aggregation of human endocrine pancreas precursor cells.

Human islet-derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC-1) cells can be induced to form aggregates that subsequently differentiate into hormone-expressing islet-like cell aggregates (ICAs). We show that challenge of hIPCs or PANC-1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol-tris-phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC-1 cells, human fetal pancreas, and human adult islets express two protease-activated receptors (PARs), PAR-1 and PAR-2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR-1 (SFLLRN-amide) or PAR-2 (SLIGRL-amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G-protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo.