Vascular Dysfunction among Malaysian Men with Increased BMI: An Indication of Synergistic Effect of Free Testosterone and Inflammation.

Background and objectives: Obesity is associated with poor vascular function and may lead to future cardiovascular disease (CVD). Obesity is also related to increased inflammation and a low testosterone level. This study was conducted to determine the relationship between inflammation, testosterone level, and vascular function among subjects with an increased body mass index (BMI) and to determine whether both low testosterone and high inflammation have synergistic effects towards vascular dysfunction. Materials and Methods: A total of 303 men aged 40-80 years were recruited from Klang Valley, Malaysia. Their height, weight, blood pressure (BP), lipid, blood glucose level, total testosterone (TT), free testosterone (FT), and C-reactive protein (CRP) were measured. The carotid femoral pulse wave velocity (PWVCF) and augmentation index (AI) were also recorded as markers of vascular function. Results: The mean age of all the subjects was 54.46 ± 9.77 years. Subjects were divided into a low/normal body mass index (BMI) group (BMI < 25 kg/m2; NG, n = 154) and high BMI group (BMI ≥ 25 kg/m2; OG, n = 149). The mean BMI for NG was 22.20 ± 1.94 kg/m2 while for OG was 28.87 ± 3.24 kg/m2 (p < 0.01). The level of TT (OG = 21.13 ± 6.44 versus NG = 16.18 ± 6.16 nmol/L, p < 0.01) and FT (OG = 0.34 ± 0.12 versus NG = 0.39 ± 0.11 nmol/L, p < 0.01) were reduced while the level of CRP [OG = 1.05 (2.80) versus NG = 0.50 (1.50) mmol/L, p = 0.01] was increased in OG compared to NG. PWVCF (OG = 8.55 ± 1.34 versus NG = 8.52 ± 1.42 m/s, p = 0.02) and AI (OG = 16.91% ± 6.00% versus 15.88% ± 5.58%, p < 0.01) were significantly increased in OG after adjustment for other CVD risk factors. The subjects that had both a low FT and an increased CRP had higher AI when compared to those with a high CRP and high FT (p < 0.01). Conclusions: The increased BMI was associated with vascular dysfunction, mediated by a low testosterone level and increased inflammation. Furthermore, having both conditions concurrently lead to higher vascular dysfunction. Weight loss, testosterone supplementation, and the anti-inflammatory agent may be beneficial for men to prevent vascular dysfunction.

Elevation of tumour markers TGF-ß, M2-PK, OV-6 and AFP in hepatocellular carcinoma (HCC)-induced rats and their suppression by microalgae Chlorella vulgaris.

BACKGROUND: Chlorella vulgaris (ChV), a unicellular green algae has been reported to have anticancer and antioxidant effects. The aim of this study was to determine the chemopreventive effect of ChV on liver cancer induced rats by determining the level and expression of several liver tumour markers. METHODS: Male Wistar rats (200-250 g) were divided into 4 groups according to the diet given: control group (normal diet), ChV group with three different doses (50, 150 and 300 mg/kg body weight), liver cancer- induced group (choline deficient diet + 0.1% ethionine in drinking water or CDE group), and the treatment group (CDE group treated with three different doses of ChV). Rats were killed at 0, 4, 8 and 12 weeks of experiment and blood and tissue samples were taken from all groups for the determination of tumour markers expression alpha-fetoprotein (AFP), transforming growth factor-ß (TGF-ß), M2-pyruvate kinase (M2-PK) and specific antigen for oval cells (OV-6). RESULTS: Serum level of TGF-ß increased significantly (p < 0.05) in CDE rats. However, ChV at all doses managed to decrease (p < 0.05) its levels to control values. Expressions of liver tumour markers AFP, TGF-ß, M2-PK and OV-6 were significantly higher (p < 0.05) in tissues of CDE rats when compared to control showing an increased number of cancer cells during hepatocarcinogenesis. ChV at all doses reduced their expressions significantly (p < 0.05). CONCLUSIONS: Chlorella vulgaris has chemopreventive effect by downregulating the expression of tumour markers M2-PK, OV-6, AFP and TGF-ß, in HCC-induced rats.

Antioxidant modulation in restoring mitochondrial function in neurodegeneration.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the leading causes of disability associated with neurodegeneration worldwide. These diseases are influenced by multiple genetic and environmental factors and share similar mechanisms as both are characterized by accumulation and aggregation of misfolded proteins - amyloid-beta (Aß) in AD and α-synuclein in PD. Over the past decade, increasing evidence has shown that mitochondrial dysfunction and the generation of reactive oxygen species (ROS) are involved in the pathology of these diseases, and the contributions of these defects to the cellular and molecular changes that eventually cause neuronal death have been explored. Using mitochondrial protective agents, such as antioxidants, to combat ROS provides a new strategy for neurodegenerative treatment. In this review, we highlight the potential of multiple types of antioxidants, including vitamins, phytochemicals, fatty acids and minerals, as well as synthetic antioxidants specifically targeting the mitochondria, which can restore mitochondrial function, in the treatment of neurodegenerative disorders at both the pre-clinical and clinical stages by focusing on AD and PD.

Acacia honey accelerates in vitro corneal ulcer wound healing model.

BACKGROUND: The study aimed to evaluate the effects of Acacia honey (AH) on the migration, differentiation and healing properties of the cultured rabbit corneal fibroblasts. METHODS: Stromal derived corneal fibroblasts from New Zealand White rabbit (n = 6) were isolated and cultured until passage 1. In vitro corneal ulcer was created using a 4 mm corneal trephine onto confluent cultures and treated with basal medium (FD), medium containing serum (FDS), with and without 0.025 % AH. Wound areas were recorded at day 0, 3 and 6 post wound creation. Genes and proteins associated with wound healing and differentiation such as aldehyde dehydrogenase (ALDH), vimentin, alpha-smooth muscle actin (α-SMA), collagen type I, lumican and matrix metalloproteinase 12 (MMP12) were evaluated using qRT-PCR and immunocytochemistry respectively. RESULTS: Cells cultured with AH-enriched FDS media achieved complete wound closure at day 6 post wound creation. The cells cultured in AH-enriched FDS media increased the expression of vimentin, collagen type I and lumican genes and decreased the ALDH, α-SMA and MMP12 gene expressions. Protein expression of ALDH, vimentin and α-SMA were in accordance with the gene expression analyses. CONCLUSION: These results demonstrated AH accelerate corneal fibroblasts migration and differentiation of the in vitro corneal ulcer model while increasing the genes and proteins associated with stromal wound healing.

The effects of acacia honey on in vitro corneal abrasion wound healing model.

BACKGROUND: Acacia honey (AH) has been proven to improve skin wound healing, but its therapeutic effects on corneal epithelium has not been elucidated to date. This study aimed to investigate the effects of AH on cultured corneal epithelial cells (CEC) on in vitro corneal abrasion wound healing model. Six New Zealand white rabbits' CEC were isolated and cultured until passage 1. Circular wound area was created onto a confluent monolayer CEC using a corneal trephine which mimicked corneal abrasion and treated with 0.025% AH supplemented in basal medium (BM) and complete cornea medium (CCM). Wound healing was measured as the percentage of wound closure by the migration of CEC on day 0, day 3 and day 6, post wound creation. The morphological changes of CEC were assessed via phase contrast microscopy. Gene and protein expressions of cytokeratin (CK3), fibronectin and cluster of differentiation 44 (CD44) in AH treated groups and control groups were determined by real-time PCR and immunocytochemistry, respectively. RESULTS: Cultured CEC exhibited similar morphology of polygonal shaped cells in all culture media. CEC cultured in AH-supplemented media showed higher percentage of wound closure compared to the controls. Gene expression of CK3 increased in AH-supplemented groups throughout the study. Fibronectin expression was increased at the initial stage while CD44 expression was increased at day 3, post wound creation. The protein expression of CEC cultured in all media was in accordance to their respective gene expressions. CONCLUSION: Supplementation of AH in BM and CCM media accelerates CEC wound closure of the in vitro corneal abrasion model by increasing the expression of genes and proteins associated with CEC wound healing.

Vitamin D is significantly associated with total testosterone and sex hormone-binding globulin in Malaysian men.

OBJECTIVE: Cross-sectional studies in the Caucasian population have shown a significant relationship between vitamin D and testosterone levels, but data in the Asian population are limited. This study aimed to determine the association between vitamin D and testosterone levels in Malaysian men. METHODS: Chinese and Malay men (n = 382) aged 20 years or above residing in the Klang Valley, Malaysia were recruited. Their fasting blood was collected for serum testosterone, sex hormone-binding globulin (SHBG) and 25-hydroxyvitamin D (25(OH)D) assays. Relationship between 25(OH)D and testosterone levels was analyzed using multiple regression analysis. Testosterone and SHBG levels among subjects with different vitamin D status were compared using univariate analysis. Confounders such as age, ethnicity and body mass index (BMI) were adjusted. RESULTS: 25(OH)D was significantly and positively associated with total testosterone and SHBG levels before and after adjustment for age and ethnicity (p < 0.05). Only association with SHBG remained significant after further adjustment for BMI (p < 0.05). Total testosterone and SHBG values displayed an increasing trend from subjects with vitamin D deficiency to those with optimal level (p < 0.05). The trend was attenuated after adjustment for BMI (p > 0.05). CONCLUSION: 25(OH)D is significantly associated with total testosterone and SHBG in Malaysian men but this association is BMI-dependent.

Gamma-tocotrienol treatment increased peroxiredoxin-4 expression in HepG2 liver cancer cell line.

BACKGROUND: To determine the antiproliferative effect of gamma-tocotrienol (GTT) treatment on differential protein expression in HepG2 cells. METHODS: HepG2 cells were treated with 70 µM GTT for 48 hours and differentially expressed protein spots were determined by two-dimensional electrophoresis (2DE), identified by MALDI-TOF mass spectrometer (MS) and validated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: GTT treatment on HepG2 cells showed a total of five differentially expressed proteins when compared to their respective untreated cells where three proteins were down-regulated and two proteins were up-regulated. One of these upregulated proteins was identified as peroxiredoxin-4 (Prx4). Validation by qRT-PCR however showed decreased expression of Prx4 mRNA in HepG2 cells following GTT treatment. CONCLUSIONS: GTT might directly influence the expression dynamics of peroxiredoxin-4 to control proliferation in liver cancer.

Insulin-like growth factor-1 is a mediator of age-related decline of bone health status in men.

OBJECTIVE: The role of insulin-like growth factor-1 (IGF-1) in bone health in men is debatable. This study aimed to determine whether IGF-1 is a mediator in age-related decline of bone health status measured by calcaneal speed of sound (SOS) in Malaysian men. METHODS: The study recruited 279 Chinese and Malay men. Their demographic data, weight, height, calcaneal SOS were taken and fasting blood was collected for total testosterone, sex-hormone binding globulin and IGF-1 assays. The associations between the studied variables were assessed using multiple linear regression (MLR) analysis. Mediator analysis was performed using Sobel test. RESULTS: There was a significant and parallel decrease of IGF-1 and SOS with age (p < 0.05). Serum IGF-1 was significantly and positively associated with SOS (p < 0.05) but after further adjustment for age, the significance was lost (p > 0.05). The strength of the association between age and SOS decreased after adjusting for IGF-1 level but it remained significant (p < 0.05). Sobel test revealed that IGF-1 was a significant partial mediator in the relationship between age and SOS (z = -4.3). CONCLUSION: Serum IGF-1 is a partial mediator in the age-related decline of bone health in men as determined by calcaneal ultrasound. A prospective study should be performed to validate this relationship.

Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells.

BACKGROUND: Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. METHODS: WT and N0 cells were treated with 5 and 10 µg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [ NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. RESULTS: Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. CONCLUSION: The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway.

Gamma-tocotrienol and hydroxy-chavicol synergistically inhibits growth and induces apoptosis of human glioma cells.

BACKGROUND: Gamma-tocotrienol (GTT), an isomer of vitamin E and hydroxy-chavicol (HC), a major bioactive compound in Piper betle, has been reported to possess anti-carcinogenic properties by modulating different cellular signaling events. One possible strategy to overcome multi-drug resistance and high toxic doses of treatment is by applying combinational therapy especially using natural bioactives in cancer treatment. METHODS: In this study, we investigated the interaction of GTT and HC and its mode of cell death on glioma cell lines. GTT or HC alone and in combination were tested for cytotoxicity on glioma cell lines 1321N1 (Grade II), SW1783 (Grade III) and LN18 (Grade IV) by [3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)- 2H- tetrazolium, inner salt] MTS assay. The interactions of each combination were evaluated by using the combination index (CI) obtained from an isobologram. RESULTS: Individually, GTT or HC displayed mild growth inhibitory effects against glioma cancer cell lines at concentration values ranging from 42-100 µg/ml and 75-119 µg/ml respectively. However, the combination of sub-lethal doses of GTT + HC dramatically enhanced the inhibition of glioma cancer cell proliferation and exhibited a strong synergistic effect on 1321N1 with CI of 0.55, and CI = 0.54 for SW1783. While in LN18 cells, moderate synergistic interaction of GTT + HC was observed with CI value of 0.73. Exposure of grade II, III and IV cells to combined treatments for 24 hours led to increased apoptosis as determined by annexin-V FITC/PI staining and caspase-3 apoptosis assay, showing caspase-3 activation of 27%, 7.1% and 79% respectively. CONCLUSION: In conclusion, combined treatments with sub-effective doses of GTT and HC resulted in synergistic inhibition of cell proliferation through the induction of apoptosis of human glioma cells in vitro.

Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes.

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits' corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.

Supplementation with tocotrienol-rich fraction alters the plasma levels of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor from young and old individuals.

PURPOSE: Tocotrienol possess beneficial effects not exhibited by tocopherol. In vitro studies using animal models have suggested that these effects are caused via modulation of gene and protein expression. However, human supplementation studies using tocotrienol-rich isomers are limited. This study aims to identify plasma proteins that changed in expression following tocotrienol-rich fraction (TRF) supplementation within two different age groups. METHODS: Subjects were divided into two age groups-32 ± 2 (young) and 52 ± 2 (old) years old. Four subjects from each group were assigned with TRF (78% tocotrienol and 22% tocopherol, 150 mg/day) or placebo capsules for 6 months. Fasting plasma were obtained at 0, 3, and 6 months. Plasma tocopherol and tocotrienol levels were determined. Plasma proteome was resolved by 2DE, and differentially expressed proteins identified by MS. The expressions of three proteins were validated by Western blotting. RESULTS: Six months of TRF supplementation significantly increased plasma levels of tocopherols and tocotrienols. Proteins identified as being differentially expressed were related to cholesterol homeostasis, acute-phase response, protease inhibitor, and immune response. The expressions of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor were validated. The old groups showed more proteins changing in expression. CONCLUSIONS: TRF appears to not only affect plasma levels of tocopherols and tocotrienols, but also the levels of plasma proteins. The identity of these proteins may provide insights into how TRF exerts its beneficial effects. They may also be potentially developed into biomarkers for the study of the effects and effectiveness of TRF supplementation.

Discrepancy between the quantitative ultrasound value of Malaysian men and the manufacturer's reference and the impact on classification of bone health status.

The local normative value in quantitative ultrasound (QUS) equipment needs to be established for wider application and accurate classification of patients into respective fracture risk groups. The present study aimed to establish the calcaneal speed of sound (SOS) value for Chinese and Malay men in Malaysia and determine the difference between calcaneal SOS of the local population and the reference values provided by the manufacturer for each age group. This study will also determine the effect of using the manufacturer's young adult (20-29yr) reference or the local young adult reference to classify the subjects into the respective risk groups. Eight hundred forty Malay and Chinese men residing in central peninsular Malaysia were recruited and their calcaneal QUS value was determined using the CM-200 machine (Furuno Electric, Nishinomiya City, Japan). The results showed that the differences in SOS values between Chinese and Malay men were not significant across all the age groups studied (p>0.05). The age-dependent reduction of SOS value assumed a biphasic form, which was evident at 30-39yr and older than 60yr. The calcaneal SOS of the subject under study was significantly higher as compared with the manufacturer's reference (based on Japanese population) in all groups aged 40yr and older (p<0.05). A significant proportion of the subjects in the osteoporosis group was misclassified using the manufacturer's young adult reference as compared with using the local young adult reference (p<0.05). In conclusion, the overall normative value of SOS obtained was suitable for Chinese and Malay men in Malaysia, and a local reference value should be applied to avoid misclassification of subjects into the respective risk groups.

Optimization of 3D Immunofluorescence Analysis and Visualization Using IMARIS and MeshLab.

The precision of colocalization analysis is enhanced by 3D and is potentially more accurate than 2D. Even though 3D improves the visualization of colocalization analysis, rendering a colocalization model may generate a model with numerous polygons. We developed a 3D colocalization model of FtMt/LC3 followed by simplification. Double immunofluorescence staining of FtMt and LC3 was conducted, and stacked images were acquired. We used IMARIS to render the 3D colocalization model of FtMt/LC3 and further processed it with MeshLab to decimate and generate a less complex colocalization model. We examined the available simplification algorithm using MeshLab in detail and evaluated the feasibility of each procedure in generating a model with less complexity. The quality of the simplified model was subsequently assessed. MeshLab's available shaders were scrutinized to facilitate the spatial colocalization determination. Finally, we showed that QECD was the most effective method for reducing the polygonal complexity of the colocalization model without compromising its quality. In addition, we would recommend implementing the x-ray shader, which we found useful for visualizing colocalization. As 3D was found to be more accurate in quantifying colocalization, our study provides a novel and dependable method for rendering 3D models for colocalization analysis.

Biomarkers for Premature Coronary Artery Disease (PCAD): A Case Control Study.

Coronary artery disease (CAD) is often associated with the older generation. However, in recent years, there is an increasing trend in the prevalence of CAD among the younger population; this is known as premature CAD. Although biomarkers for CAD have been established, there are limited studies focusing on premature CAD especially among the Malay male population. Thus, the aim of this research was to compare the biomarkers between premature CAD (PCAD) and older CAD (OCAD) among Malay males. Subjects, recruited from the Universiti Kebangsaan Malaysia Medical Centre and National Heart Institution, were divided into four groups: healthy control < 45 years old; premature CAD (PCAD) < 45 years old; healthy control > 60 years old; and older CAD (OCAD) > 60 years old, with n = 30 for each group. Ten potential markers for CAD including soluble sVCAM-1, sICAM-1, interleukin-2, interleukin-6, interleukin-10, Apo-E and Apo-A1, homocysteine, CRP, and vitamin D levels were examined. Our results revealed premature CAD patients had significantly higher values (p < 0.05) of sVCAM-1, CRP, interleukin-6, and vitamin D when compared to the age-matched controls. Similarly, older CAD patients showed higher levels of sVCAM-1, CRP, and interleukin-2 when compared to their age-matched controls. After adjusting for multiple parameters, only CRP remained significant for PCAD and interleukin-2 remained significant for CAD. This indicates that premature CAD and older CAD patients showed different profiles of protein biomarkers. CRP has the potential to become a biomarker for premature CAD while interleukin-2 is a better biomarker for older CAD together with other typical panels of protein biomarkers.

Metabolomics Profiling of Age-Associated Metabolites in Malay Population.

Aging is a complex process characterized by progressive loss of functional abilities due to the accumulation of molecular damages. Metabolomics could offer novel insights into the predictors and mechanisms of aging. This cross-sectional study is aimed at identifying age-associated plasma metabolome in a Malay population. A total of 146 (90 females) healthy participants aged 28-69 were selected for the study. Untargeted metabolomics profiling was performed using liquid chromatography-tandem mass spectrometry. Association analysis was based on the general linear model. Gender-associated metabolites were adjusted for age, while age-associated metabolites were adjusted for gender or analyzed in a gender-stratified manner. Gender-associated metabolites such as 4-hydroxyphenyllactic acid, carnitine, cortisol, and testosterone sulfate showed higher levels in males than females. Deoxycholic acid and hippuric acid were among the metabolites with a positive association with age after being adjusted for gender, while 9(E),11(E)-conjugated linoleic acid, cortisol, and nicotinamide were negatively associated with age. In gender-stratified analysis, glutamine was one of the common metabolites that showed a direct association with age in both genders, while metabolites such as 11-deoxy prostaglandin F2ß, guanosine monophosphate, and testosterone sulfate were inversely associated with age in males and females. This study reveals several age-associated metabolites in Malays that could reflect the changes in metabolisms during aging and may be used to discern the risk of geriatric syndromes and disorders later. Further studies are required to determine the interplay between these metabolites and environmental factors on the functional outcomes during aging.

Missense mutations in MLH1, MSH2, KRAS, and APC genes in colorectal cancer patients in Malaysia.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide with approximately 1 million cases diagnosed annually. In Malaysia, CRC is the second most common cancer in women and ranked first in men. The underlying cause of CRC remains unknown. AIMS: The aim of this study was to analyze the mutations in genes involved in CRC including MLH1, MSH2, KRAS, and APC genes. METHODS: A total of 76 patients were recruited. We used the polymerase chain reaction-denaturing high-performance liquid chromatography for the detection of mutations in the mismatch repair (MMR) and APC genes and the PCR single-strand conformation polymorphism for screening of the KRAS gene mutations. RESULTS: We identified 17 types of missense mutations in 38 out of 76 patients in our patients. Nine mutations were identified in the APC gene, five mutations were detected in the KRAS gene, and two mutations were identified in the MSH2 gene. Only one mutation was identified in MLH1. Out of these 17 mutations, eight mutations (47 %) were predicted to be pathogenic. Seven patients were identified with multiple mutations (3: MSH2 and KRAS, 1: KRAS and APC, 1: MLH1 and APC, 2: APC and APC). CONCLUSIONS: We have established the PCR-DHPLC and PCR-SSCP for screening of mutations in CRC patients. This study has given a snapshot of the spectrum of mutations in the four genes that were analyzed. Mutation screening in patients and their family members will help in the early detection of CRC and hence will reduce mortality due to CRC.

Evaluation of topical tocopherol cream on cutaneous wound healing in streptozotocin-induced diabetic rats.

Diabetes is a common cause of delayed wound healing. The aim of the study was to determine the effect of topical administration of tocopherol cream on the wound healing process in diabetic rats. The study was conducted using 18 male Sprague Dawley rats which were divided into three groups: (I) diabetic rats receiving control cream (n = 6), (II) diabetic rats receiving 0.06% tocopherol cream (n = 6), and (III) diabetic rats receiving 0.29% tocopherol cream (n = 6). Four cutaneous wounds were created at the dorsal region of the rats. Wound healing was assessed by total protein content, rate of wound closure estimation, and histological studies on the tenth day after wounding. Tocopherol treatment enhanced the wound healing process by increasing rate of wound closure and total protein content significantly (P < 0.05) compared to the control group. Histological observation also showed better organized epithelium and more collagen fibers in the tocopherol treated groups. Application of tocopherol cream enhances wound healing process in diabetic condition which is known to cause delay in wound healing.

Inhibitory Mechanism of Combined Hydroxychavicol With Epigallocatechin-3-Gallate Against Glioma Cancer Cell Lines: A Transcriptomic Analysis.

Emerging reports have shown therapeutic potential of hydroxychavicol (HC) and epigallocatechin-3-gallate (EGCG) against cancer cells, however high concentrations are required to achieve the anticancer activity. We reported the synergy of low combination doses of EGCG+HC in glioma cell lines 1321N1, SW1783, and LN18 by assessing the effects of EGCG+HC through functional assays. Using high throughput RNA sequencing, the molecular mechanisms of EGCG+HC against glioma cell lines were revealed. EGCG/HC alone inhibited the proliferation of glioma cell lines, with IC50 values ranging from 82 to 302 µg/ml and 75 to 119 µg/ml, respectively. Sub-effective concentrations of combined EGCG+HC enhanced the suppression of glioma cell growth, with SW1783 showing strong synergism with a combination index (CI) of 0.55 and LN18 showing a CI of 0.51. A moderate synergistic interaction of EGCG+HC was detected in 1321N1 cells, with a CI value of 0.88. Exposure of 1321N1, SW1783, and LN18 cells to EGCG+HC for 24 h induces cell death, with caspase-3 activation rates of 52%, 57%, and 9.4%, respectively. However, the dose for SW1783 is cytotoxic to normal cells, thus this dose was excluded from other tests. EGCG+HC induced cell cycle arrest at S phase and reduced 1321N1 and LN18 cell migration and invasion. Combined EGCG+HC amplified its anticancer effect by downregulating the axon guidance process and metabolic pathways, while simultaneously interfering with endoplasmic reticulum unfolded protein response pathway. Furthermore, EGCG+HC exerted its apoptotic effect through the alteration of mitochondrial genes such as MT-CO3 and MT-RNR2 in 1321N1 and LN18 cells respectively. EGCG+HC dynamically altered DYNLL1 alternative splicing expression in 1321N1 and DLD splicing expression in LN18 cell lines. Our work indicated the pleiotropic effects of EGCG+HC treatment, as well as particular target genes that might be investigated for future glioma cancer therapeutic development.

Alpha- and Gamma-Tocopherol Modulates the Amyloidogenic Pathway of Amyloid Precursor Protein in an in vitro Model of Alzheimer's Disease: A Transcriptional Study.

The amyloid precursor protein (APP) processing pathway was altered in Alzheimer's disease (AD) and contributed to abnormal amyloid-beta (Aß) production, which forms insoluble interneuron protein aggregates known as amyloid plaques in the brain. Targeting the APP processing pathway is still fundamental for AD modifying therapy. Extensive research has evaluated the protective effects of vitamin E as an antioxidant and as a signaling molecule. The present study aimed to investigate the modulatory effects of different tocopherol isomers on the expression of genes involved in regulating the APP processing pathway in vitro. The screening for the effective tocopherol isomers in reducing APP expression and Aß-42 was carried out in SH-SY5Y stably overexpressed APP Swedish. Subsequently, quantitative one-step real-time PCR was performed to determine the modulatory effects of selected tocopherol isomers on the expression of genes in SH-SY5Y stably overexpressed three different types of APP (wild-type, APP Swedish, and APP Swedish/Indiana). Our results showed that all tocopherol isomers, especially at higher concentrations (80-100 µM), significantly increased (p < 0.05) the cell viability in all cells group, but only α-tocopherol (ATF) and γ-tocopherol (GTF) significantly decreased (p < 0.05) the APP mRNA level without statistically significant APP protein level, accompanied with a reduced significance (p < 0.05) on the level of Aß-42 in SH-SY5Y APP Swedish. On the other hand, ß- and δ-tocopherol (BTF and DTF) showed no effects on the level of APP expression and Aß-42. Subsequent results demonstrated that ATF and GTF significantly decreased (p < 0.05) the expression of gene beta-site APP cleaving enzyme (BACE1), APH1B, and Nicastrin (NCSTN), but significantly increased (p < 0.05) the expression of Sirtuin 1 (SIRT1) in SH-SY5Y stably expressed the mutant APP form. These findings suggested that ATF and GTF could modulate altered pathways and may help ameliorate the burden of amyloid load in AD.