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M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 μM) and ERK kinase (MEK) inhibitor PD98059 (20 μM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 μM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.

Background: Gastrointestinal (GI) mucosal injury is a frequent complication of long-term nonsteroidal anti-inflammatory drug (NSAID) use. Effective mucosal healing requires coordinated epithelial migration, proliferation, and angiogenesis, which may be influenced by focal adhesion kinase (FAK). This study aimed to determine whether our newly developed FAK activators promote intestinal mucosal healing by enhancing angiogenesis and whether FAK activation increases resistance to reinjury. Methods: Ischemic jejunal ulcers were induced in C57BL/6 mice. After 24 h, mice received intraperitoneal injections of the FAK activator ZINC40099027 (ZN27, 900 µg/kg every 6 h) or vehicle for 2, 4, or 14 days. Ulcer areas were quantified, and liver and kidney function were assessed. Ulcer and adjacent tissues were analyzed by immunofluorescence staining for angiogenesis and proliferation markers. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with ZN27 to evaluate proliferation, migration, angiogenesis, and intracellular signaling. In a reinjury model, male C57BL/6J mice received continuous infusion of the FAK activator M64HCl (25 mg/kg/day) or vehicle for 7 days, with a single subcutaneous injection of indomethacin (10 mg/kg) on day 1 to induce GI injury. Fourteen days after the first dose of indomethacin, the mice received a second indomethacin challenge, and one day later, total ulcer areas in the pyloric opening and small intestine were quantified. Results: Ulcer areas were significantly smaller in ZN27-treated mice compared with vehicle-treated controls at 3 and 5 days, accompanied by increased expression of angiogenesis and proliferation markers. In vitro, ZN27 enhanced HUVEC migration via FAK activation in an ERK1/2-dependent manner and increased the number of angiogenic sprouts. In the reinjury model, treatment with M64HCl during the initial indomethacin-induced injury resulted in significantly smaller ulcer areas in both the pyloric opening and small intestine after the second indomethacin challenge compared with controls. Conclusions: FAK activation accelerates ischemic ulcer healing, in part by enhancing angiogenesis. Moreover, FAK activation during an initial injury reduces susceptibility to recurrent NSAID-induced intestinal injury, perhaps because it promotes initial higher-quality ulcer repair.

Nonsteroidal anti-inflammatory drugs cause gastric ulcers and gastritis. No drug that treats GI injury directly stimulates mucosal healing. ZINC40099027 (ZN27) activates focal adhesion kinase (FAK) and heals acute indomethacin-induced small bowel injury. We investigated the efficacy of ZN27 in rat and human gastric epithelial cells and ongoing aspirin-associated gastric injury. ZN27 (10 nM) stimulated FAK activation and wound closure in rat and human gastric cell lines. C57BL/6J mice were treated with 300 mg/kg/day aspirin for five days to induce ongoing gastric injury. One day after the initial injury, mice received 900 µg/kg/6 h ZN27, 10 mg/kg/day omeprazole, or 900 µg/kg/6 h ZN27 plus 10 mg/kg/day omeprazole. Like omeprazole, ZN27 reduced gastric injury vs. vehicle controls. ZN27-treated mice displayed better gastric architecture, with thicker mucosa and less hyperemia, inflammation, and submucosal edema, and lost less weight than vehicle controls. Gastric pH, serum creatinine, serum alanine aminotransferase (ALT), and renal and hepatic histology were unaffected by ZN27. Blinded scoring of pFAK-Y-397 immunoreactivity at the edge of ZN27-treated lesions demonstrated increased FAK activation, compared to vehicle-treated lesions, confirming target activation in vivo. These results suggest that ZN27 ameliorates ongoing aspirin-associated gastric mucosal injury by a pathway involving FAK activation. ZN27-derivatives may be useful to promote gastric mucosal repair.

ZINC40099027 (ZN27) is a specific focal adhesion kinase (FAK) activator that promotes murine mucosal wound closure after ischemic or NSAID-induced injury. Diverse motogenic pathways involve FAK, but the direct consequences of pure FAK activation have not been studied, and how ZN27-induced FAK activation stimulates wound closure remained unclear. We investigated signaling and focal adhesion (FA) turnover after FAK activation by ZN27 in Caco-2 cells, confirming key results in CCD841 cells. ZN27 increased Caco-2 FAK-Y-397, FAK-Y-576/7, paxillin-Y-118, and ERK 1/2 phosphorylation and decreased FAK-Y-925 phosphorylation, without altering FAK-Y-861, p38, Jnk, or Akt phosphorylation. ZN27 increased FAK-paxillin interaction while decreasing FAK-Grb2 association. ZN27 increased membrane-associated FAK-Y-397 and FAK-Y-576/7 phosphorylation and paxillin-Y-118 and ERK 1/2 phosphorylation but decreased FAK-Y-925 phosphorylation without altering Src or Grb2. Moreover, ZN27 increased the fluorescence intensity of GFP-FAK and pFAK-Y397 in FAs and increased the total number of FAs but reduced their size in GFP-FAK-transfected Caco-2 cells, consistent with increased FA turnover. In contrast, FAK-Y397F transfection prevented ZN27 effects on FAK size and number and FAK and pFAK fluorescent intensity in FAs. We confirmed the proposed FAK/paxillin/ERK pathway using PP2 and U0126 to block Src and MEK1/2 in Caco-2 and CCD841 cells. These results suggest that ZN27 promotes intestinal epithelial monolayer defect closure by stimulating autophosphorylation of FAK in the cytosol, distinct from classical models of FAK activation in the FA. Phosphorylated FAK translocates to the membrane, where its downstream substrates paxillin and ERK are phosphorylated, leading to FA turnover and human intestinal epithelial cell migration. Graphical abstract

Of those, three used cluster sampling,3-5 one intended to reach the entire population living in the study area20 and one did not mention how the sampling procedure was managed.19 Four of those studies were held mainly in coastal urban areas,3-5,19 not taking into consideration the hinterland and rural areas. [...]our study had the advantage of covering both urban and rural areas and also inland Turkey. In doing so, we aimed to capture all the individuals intended to be involved in the study. [...]despite being less costly than face-to-face surveys, telephone surveys might result in lower response rates.21 Our study indicates a prevalence of 0.56% for RA adjusted for the general population aged 16 or over. [...]58% of the individuals involved in our study were females whereas the national census performed in 2000 showed that females constituted 50% of Turkey's population. [...]our study showed that a northsouth gradient of RA across Europe might not exist, compared to the prevalence reported from Northern European countries.12,28 However, a north-south gradient of RA might exist in Turkey.

Increasing dietary fiber consumption is linked to lower colon cancer incidence, and this anticancer effect is tied to elevated levels of short-chain fatty acids (e.g., butyrate) because of the fermentation of fiber by colonic bacteria. While butyrate inhibits cancer cell proliferation, the impact on cancer cell type remains largely unknown. To test the hypothesis that butyrate displays different inhibitory potentials due to cancer cell type, we determined half-maximal inhibitory concentrations (IC50) of butyrate in HCT116, HT-29, and Caco-2 human colon cancer cell proliferation at 24, 48, and 72 h. The IC50 (mM) butyrate concentrations of HCT116, HT-29, and Caco-2 cells were [24 h, 1.14; 48 h, 0.83; 72 h, 0.86], [24 h, N/D; 48 h, 2.42; 72 h, 2.15], and [24 h, N/D; 48 h, N/D; 72 h, 2.15], respectively. At the molecular level, phosphorylated ERK1/2 and c-Myc survival signals were decreased by (>30%) in HCT116, HT-29, and Caco-2 cells treated with 4 mM butyrate. Conversely, butyrate displayed a stronger potential (>1-fold) for inducing apoptosis and nuclear p21 tumor suppressor in HCT116 cells compared to HT-29 and Caco-2 cells. Moreover, survival analysis demonstrated that a cohort with high p21 gene expression in their colon tissue significantly increased survival time compared to a low-p21-expression cohort of colon cancer patients. Collectively, the inhibitory efficacy of butyrate is cell type-specific and apoptosis-dependent.

Gastrointestinal (GI) ulcers are common worldwide. The persistence of noxious agents such as gastric acid, pepsin, nonsteroidal anti-inflammatory drugs (NSAIDs), and inflammation caused by inflammatory bowel disease breaks down the mucosal barrier and causes mucosal injury in the GI tract. Currently available therapeutics such as proton pump inhibitors (PPIs), histamine-2 receptor antagonists (H2-antagonists), antibiotics, and corticosteroids all attempt to minimize injury by reducing the symptoms of GI pathology, however, they do not directly promote mucosal healing. NSAIDs increase the development of both upper and lower GI ulcers. PPIs ameliorate upper GI injury; however, PPIs actually worsen NSAID distal enteropathy by suppressing gastric acid secretion and changing the enteric microbiome. Therefore, there is an urgent need for a therapeutic agent that directly promotes both upper and lower GI healing. The non-receptor tyrosine kinase Focal Adhesion Kinase (FAK), 125 kDa, regulates the maturation and turnover of focal adhesion (FA) and thus, cell migration. We developed a series of small molecule FAK activators such as M64HCl, a derivative of a small molecule called ZINC40099027 (ZN27). To investigate the effects of these FAK activators, we utilized western blot, monolayer wound closure assays, co-immunoprecipitation, subcellular fractionation assays, and Immunofluorescent staining in a series of gastrointestinal epithelial cells and we used an ongoing aspirin-induced gastric ulcer model in vivo. In chapter one, we reviewed the importance of gut homeostasis, physiology of mucosal healing, protective factors for the gastrointestinal mucosa, drivers of mucosal injury, mucosal healing processes, restitution and quality of ulcer healing, regulators of mucosal healing, and potential new therapeutic targets. In chapter two, we, first, demonstrated that ZN27 (10 nM) stimulated the activation of FAK and monolayer wound closure in rat and human gastric cell lines. Then, in vivo studies revealed that four days of 900µg/kg/6h ZN27 ameliorates gastric injury similar to 10 mg/kg/day omeprazole positive control vs. vehicle controls in C57BL/6J mice treated with 300mg/kg/day aspirin for five days to induce ongoing gastric injury, without obvious toxicity, by a pathway involving FAK activation. In chapter three, we displayed that ZN27 increases intestinal epithelial monolayer wound closure by stimulating the autophosphorylation of FAK in the cytosol, distinct from long-established models of FAK activation. Its downstream substrates paxillin and ERK are phosphorylated right after phosphorylated FAK translocates to the membrane, leading to FA turnover and human intestinal epithelial cell migration. In chapter four, we demonstrated that once three-hour or twice two-hour treatment of M64HCl (100 nM) promotes sustained monolayer wound closure by stimulating prolonged activation of FAK and ERK1/2, suggesting that increasing its half-life to 2 to 3 hours might be beneficial to promote gastrointestinal mucosal healing in clinical settings. In conclusion, small molecule FAK activators showed promising results toward gut mucosal healing. M64HCl, specifically, is a promising novel compound with water-soluble and drug-like properties. Hence, designing and optimizing the next-generation small molecule with a longer half-life might be beneficial to ameliorate both upper and lower GI mucosal injuries.

BACKGROUND/AIMSThis study aimed to determine the effects of back school therapy, a home exercise program, and shortwave diathermy treatment on the quality of life of patients with chronic low back pain.MATERIAL and METHODSIn this interventional study, we evaluated 90 patients who were admitted to our clinic from 2006 to 2007 and had been followed-up for their low back pain complaints that had been present for at least 6 months. These patients were randomized into 3 groups after being subjected to back school therapy and home exercise programs. Placebo shortwave, continuous shortwave diathermy, and pulsed shortwave diathermy treatments were applied to these three groups, respectively. The Short form 36 (SF-36) was used to evaluate the quality of life of the study groups.RESULTSIn this study, statistically significant recovery was achieved in all the three groups in terms of physical function, role limitations due to physical problems, and bodily pain criteria. Those who received the diathermy therapy had higher mean scores in terms of all the three criteria mentioned above.CONCLUSIONSignificant improvements were achieved within the study groups in terms of all the above-mentioned criteria, except for “role limitations due to emotional problems”, as measured by SF-36. However, we could not find any significant difference between the groups in terms of the quality of life.

Objective: The aim of this study was to compare exercise, continuous short-wave diathermy (Swój and intermittent SWD treatment modalities and to evaluate the effects of these treatments on chronic back pain and depression. Methods: This study is an intervention trial which evaluated patients who applied to our clinic due to chronic low back pain between 2008-2009. The study group consisted of 90 patients between the ages of 40-65 who had had low back pain for more than 6 months. The patients were randomized into three groups. The first group received placebo short-wave diathermy, the second group received continuous short-wave diathermy, and the third group received pulsed short-wave diathermy. Pain was evaluated by Visual Analog Scale (VAS) and the Pain Disability Index (PDI).The Modified Oswestry Low Back Pain Disability Questionnaire Form was used for the measurement of functional deficiency and the Beck Depression Inventory (BDI) was used for the evaluation of depression.All scales were performed before the treatment, immediately after treatment and 3 months after treatment. Results: Significant decreases in PDI and VAS scores were found in all groups (p<0.05 for each). Similarly, there was a significant improvement in all groups in terms of functional deficiency(p<0.001 for each), while no differences were found between groups (p = 0.895). In terms of BDI scores, there was no improvement in those receiving only exercise, while Group 2 and 3 had significant improvements (p <0.05). When groups were compared for BDI scores, no differences were found between any of the groups (p = 0.189). Conclusion: Continuous SWD treatment with exercise was found to be more effective in reducing pain in patients with chronic low back pain than other treatment modalities used in our study. Although there was no significant difference between the groups in terms of depressive mood, it was found that those receiving continuous and pulsed SWD treatment had significant improvements in depression as measured by the BDI.

The purpose of this study was to investigate the associations of tooth loss with skeletal bone mass, years since menopause, educational level, current smoking status, dietary calcium intake, and number of pregnancies in postmenopausal osteoporotic women in Turkey. The study population consisted of 1171 postmenopausal women aged 40-86 years (mean age, 61.19 +/- 7.28 years). A detailed history was obtained from all women, including relevant lifestyle parameters, risk factors, and measurements of weight and height. Women were separated into three groups according to the number of teeth remaining as group 1 (edentulous, 457 women), group 2 (10 or fewer teeth, 232 women), and group 3 (more than 10 teeth remaining, 482 women). There was no significant difference among the three groups in mean age and menopausal age ( P < 0.05). Body mass index of group 1 was significantly higher than that of group 2 ( P< 0.01). Educational level was significantly different between three groups: groups 1 and 2 ( P< 0.001), groups 1 and 3 ( P< 0.0001), and groups 2 and 3 ( P< 0.001). Educational level was lowest in group 1 and highest in group 3. Despite a low ratio of cigarette smoking in general, a smoking habit was most prevalent in group 3 and least in group 2. The ratio of women receiving adequate calcium was significantly lower in group 1 than in other groups ( P< 0.001); mean calcium intake was similar in all groups. The number of pregnancies was significantly higher in group 1 than in other groups ( P< 0.001). Lumbar bone mineral density (BMD) of group 1 was significantly lower than that of groups 2 and 3 ( P< 0.001). Although no significant difference was found between groups 1 and 3, femoral neck BMD of group 2 was less than in others, and differences between groups 1 and 2 and between groups 2 and 3 ( P< 0.001) were significant. Lumbar bone mineral content (BMC) of group 1 was significantly lower than that of groups 2 and 3 ( P< 0.001), and lumbar BMC in group 2 was significantly higher than in group 3 ( P< 0.05). Femoral neck BMC in group 1 was significantly higher than in groups 2 and 3 ( P< 0.001). In conclusion, lumbar BMD and BMC in the edentulous group were significantly lower, whereas femoral neck BMD and BMC were significantly higher in edentulous group compared with the others. Our findings indicated that improvement in lifestyle factors and nutritional strategies for the treatment and prevention of osteoporosis may have additional benefit in reducing tooth loss.