Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia.

BACKGROUND: Although E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection. METHODS: The core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab-) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab+) samples that included viremic (chronic/relapsers) and recovered patients' sera. The color intensity (OD450) and S/Co values were estimated. RESULTS: The integration of 0.1-0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab-sera. The ideal S/Co threshold predicting active viremia (> 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection. CONCLUSION: The applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings.

Changes in Rat Adrenal Cortex and Pineal Gland in Inverted Light-Dark Cycle: A Biochemical, Histological, and Immunohistochemical Study.

Poor sleep standards are common in everyday life; it is frequently linked to a rise in stress levels. The adrenal gland interacts physiologically with the pineal gland in the stress response. Pineal gland is a small endocrine organ that modulates sleep patterns. This work aimed to evaluate the inverted light-dark cycle rhythm on the histological changes within the adrenal cortex and pineal gland in adult male albino rats. Twenty adult male albino rats were equally divided into two groups: For the first control group, animals were kept on daylight-darkness for 12-12 h. The second group was kept under an inverted 12- to 12-h light-darkness cycle for 4 weeks. Adrenal sections were subjected to biochemical, histological, and immunohistochemical study. Inverted light-dark cycle group recorded a significant elevation of plasma corticosterone, tissue malondialdehyde, tumor necrosis factor-α, and interleukin-1ß (IL-1ß) associated with a significant reduction of catalase and superoxide dismutase. Adrenal cortex showed biochemical and histological changes. Pineal glands also showed loss of lobular architecture. A significant upregulation in activated inducible nitric oxide synthase (iNOS) and B-cell lymphoma-associated X (Bax) immunohistochemical expression was recorded in adrenal cortex associating with downregulation in B-cell lymphoma 2 (Bcl-2). It could be concluded that subchronic inverted light-dark cycle exerted direct effects on adrenal cortex and the pineal glands.

Toxicological profiling of a de novo synthesized benzimidazole derivative with potent and selective proapoptotic potentials against breast cancer.

This study aimed to investigate the toxicological profile of 1-(6-(1H-benzo[d]imidazole-2-yl)-2-methylpyridin-3-yl) ethanone (BMPE), both in vitro and in vivo. The proapoptotic/necrotic and cell cycle arrest potentials of BMPE were assessed in MCF-7 cell line. The in vivo toxicology was assessed in female Balb/c mice by repeated dosing of 5, 25, and 50 mg/kg for 21 consecutive days, then different biochemical, inflammatory, and oxidative markers were assessed in sera/tissue homogenates of treated animals. The new derivative showed a potent selective cytotoxicity against malignant cell lines with IC50 value 0.2 µM/mL, while the cytotoxic effect on normal Wi-38 cells was observed at IC50 value 0.4 µM/mL; i.e. twofold the effective anticancer dose. BMPE exhibited an early DNA fragmentation-derived cell apoptosis observed at the G0/G1 checkpoint. In vivo, BMPE was biochemically/immunologically tolerable at a pharmacological dose range of 5-25 mg/kg, with no significant rates of mortality/morbidity and minimal-to-moderate histopathological alterations recorded. The new derivative represents an attractive therapeutic candidate for breast cancer, considering its noticeable modulatory effect on the oxidative-inflammatory axis that would relate to its potent antitumor effect.

Prebiotic-mediated gastroprotective potentials of three bacterial levans through NF-κB-modulation and upregulation of systemic IL-17A.

This study aimed to investigate whether the gastroprotective effects of three types of bacterial levans are correlated with their prebiotic-associated anti-inflammatory/antioxidant potentials. Three levans designated as LevAE, LevP, and LevZ were prepared from bacterial honey isolates; purified, and characterized using TLC, NMR, and FTIR. The anti-inflammatory properties of levan preparations were assessed in LPS-stimulated RAW 264.7 cell lines, while their safety and gastroprotective potentials were assessed in Wistar rats. The three levans significantly reduced ulcer number (22.29-70.05 %) and severity (31.76-80.54 %) in the ethanol-induced gastric ulcer model compared to the control (P < 0.0001/each), with the highest effect observed in LevAE and levZ (200 mg/each) (P < 0.0001). LevZ produced the highest levels of glutathione; catalase activity, and the lowest MDA levels (P = 0.0001/each). The highest anti-inflammatory activity was observed in LevAE and levZ in terms of higher inhibitory effect on IL-1ß and TNF-α production (P < 0.0001 each); COX2, PGE2, and NF-κB gene expression. The three levan preparations also proved safe with no signs of toxicity, with anti-lipidemic properties as well as promising prebiotic activity that directly correlated with their antiulcer effect. This novel study highlights the implication of prebiotic-mediated systemic immunomodulation exhibited by bacterial levans that directly correlated with their gastroprotective activity.

Myricitrin: A promising herbal therapy for periodontitis in immunosuppressed status.

Background: Periodontitis is a complex chronic inflammatory disease aggravated in immunosuppressed patients. However, adjuvant therapies can alleviate severe inflammation and slow down disease progression. Objective: To evaluate the efficacy of myricitrin, a herbal flavonoid glycoside, in reducing immunosuppression-associated periodontitis and compare its effects with that of alendronate on alveolar bone regeneration. Methods: Fifty albino Wistar rats were randomly allocated to the control, periodontitis, immunosuppressant, myricitrin, and alendronate groups. Ligature-associated periodontitis was induced in all groups, except the control group. Cyclosporin A (CsA) was administered subcutaneously in the immunosuppressant group for immunosuppression. The myricitrin group received CsA and myricitrin, whereas the alendronate group received CsA and alendronate. The therapeutic efficacies of myricitrin and alendronate were compared histologically, morphometrically, and biochemically. Results: Myricitrin reversed bone destruction in the periodontitis and immunosuppressant groups. Morphometrically, myricitrin showed comparable improvements to alendronate in terms of gaining more bone area to 49.4 ± 4.6 and 59.5 ± 2%, respectively (P < 0.001 in relation to the untreated periodontitis group). Concomitantly, myricitrin increased osteoblast count significantly to 28.4 ± 4.7 closer to the 34.5 ± 2.4 count in the alendronate group (P < 0.001 compared with 22.5 ± 2.6 count of the immunosuppressant group). Moreover, myricitrin restored the serum calcium to 9.4 ± 0.6 mg/dL and alkaline phosphatase up to 112.9 ± 2.9 IU/L, which were almost normal levels similar to the control cohort (P > 0.05). Conclusion: Myricitrin showed beneficial effects in counteracting bone resorption in subjects with immunosuppression-associated periodontitis. Its efficacy in slowing down disease progression was comparable to that of alendronate.

Novel naringin tablet formulations of agro-resides based nano/micro crystalline cellulose with neuroprotective and Alzheimer ameliorative potentials.

This study aimed to prepare micro/nanocrystalline cellulose-loaded naringin (NAR) tablets and evaluate their neuro-protective/therapeutic potentials in Alzheimer's disease (AD) model. Micro/nanocellulose was prepared from different agro-wastes, and the different cellulose preparations were then used to formulate eight oral tablets of naringin micro/nanoparticles by direct compression. AD-like symptoms were induced in adult male Sprague Dawley rats by co-administration of 150 mg/kg AlCl3 and 300 mg/kg D-galactose (oral administration/one week), and NAR tablets were assessed for neuroprotective/therapeutic potentials in terms of behavioral changes, levels of neurodegenerative and inflammatory markers, brain redox status, neurotransmitter tones, and cortex/hippocampus histopathological alterations. NAR treatments have significantly reversed the neurotoxic effect of AlCl3 as demonstrated by improved spatial and cognitive memory functions and promoted antioxidant defense mechanisms in treated AD animals. Also, the neurodegeneration was markedly restrained as reflected by marked histopathological enhancement, and prevention/amelioration of neuropsychiatric disorders, besides the restorative effect on dysregulated neurotransmitters tone. Both NAR tablet forms showed an overall higher ameliorative effect compared to the DPZ reference drug. The formulated tablets represent promising neuroprotective/therapeutic agents for Alzheimer's disease.

Prevalence of oral lesions and evaluation of the periodontal status among diabetic females attending Princess Nourah Bint Abdulrahman University (PNU).

Objective: To evaluate the prevalence of different oral lesions and periodontal status among diabetic Saudi female patients in the College of Dentistry, PNU University. Materials and Method: A retrospective study was performed by reviewing the files of all patients who visited the College of Dentistry, PNU University, during the last 5 years. We selected diabetic 20-40-year-old Saudi female patients. Ethical approval was obtained from the Institutional Review Board of PNU University. Data collection sheets were used to gather information on demographics, education, medical and dental history, and extra and intra-oral findings. Data on the duration of diabetes, any major complications, and type of diabetes therapy were retrieved from medical records. The data were entered into an Excel sheet, and descriptive statistics were performed. The analytical phase proceeded to correlate oral lesions with patient age, type of diabetes, and periodontal status. Result: A total of 226 diabetic patients were found after reviewing the records. The most common oral mucosal lesions were traumatic ulcers (10.2%), cheek biting (8.8%), and fissured tongue (8.4%). Furthermore, 81.86% of them had periodontitis, and 18.14% had a healthy periodontal status. Conclusion: The prevalence of periodontitis among diabetic female patients is higher than that of oral lesions. Stage III periodontitis showed the strongest association to oral lesion.

Solvent polarity dictates the anti-inflammatory potency and mechanism of two purslane (Portulaca oleracea) seed extracts.

This study aimed to assess the effect of solvent polarity on anti-inflammatory potency and the underlying mechanisms of two purslane seed extracts. Methanol and dichloromethane extracts were prepared using Soxhlet extraction and chromatographically analyzed. Antioxidant activities were assessed by different assays, while the anti-inflammatory potentials were assessed in RAW 264.7 macrophage cells. Methanol extraction yielded 15.5% water-soluble extract while dichloromethane produced 3.74% fixed oil. Nineteen phenolic compounds were chromatographically identified in methanol extract compared with 16 in the fixed oil including omega fatty acids and phytosterols. Methanol extract showed significantly higher capacity in radical scavenging assays (p < .001), but the fixed oil showed higher total antioxidant capacity (p < .001). Both extracts demonstrated anti-inflammatory potentials with different mechanisms, where the phenol-rich methanol extract significantly reduced TNF-α (p = .0371) and IL-1ß (p = .0029) production through an antioxidant-mediated pathway, while the fixed oil inhibited COX1, COX2, and PGE2 gene expression through the upregulation of IL-10. PRACTICAL APPLICATIONS: Both purslane extracts presented herein demonstrated remarkable antioxidant/ anti-inflammatory potentials that could be safely utilized as natural antioxidants and inflammation remedies or as functional food products, particularly that they showed no cytotoxic effects.

Stable, efficient, and cost-effective system for the biosynthesis of recombinant bacterial cellulose in Escherichia coli DH5α platform.

BACKGROUND: Owing to its remarkable mechanical properties that surpass the plant-based cellulose, bacterial cellulose production has been targeted for commercialization during the last few years. However, the large-scale production of cellulose is generally limited by the slow growth of producing strains and low productivity which ultimately makes the commercial production of cellulose using the conventional strains non cost-effective. In this study, we developed a novel plasmid-based expression system for the biosynthesis of cellulose in E. coli DH5α and assessed the cellulose productivity relative to the typically used E. coli BL21 (DE) expression strain. RESULTS: No production was detected in BL21 (DE3) cultures upon expression induction; however, cellulose was detected in E. coli DH5α as early as 1 h post-induction. The total yield in induced DH5α cultures was estimated as 200 ± 5.42 mg/L (dry weight) after 18 h induction, which surpassed the yield reported in previous studies and even the wild-type Gluconacetobacter xylinum BRC5 under the same conditions. As confirmed with electron microscope micrograph, E. coli DH5α produced dense cellulose fibers with ~ 10 µm diameter and 1000-3000 µm length, which were remarkably larger and more crystalline than that typically produced by G. hansenii. CONCLUSIONS: This is the first report on the successful cellulose production in E. coli DH5α which is typically used for plasmid multiplication rather than protein expression, without the need to co-express cmcax and ccpAx regulator genes present in the wild-type genome upstream the bcs-operon, and reportedly essential for the biosynthesis.

Urinary transferrin and proinflammatory markers predict the earliest diabetic nephropathy onset.

AIM: This study aimed to determine the earliest markers of diabetic nephropathy (DN) onset with discriminative potentials from controlled diabetes (CD). METHODS: Sixty male Wistar rats were allocated into three groups (20/group), the two diabetic groups CD and DN received 45 and 65 mg/kg STZ in 0.1 mole/L citrate buffer, respectively, while the control group received only the vehicle. Serum/urinary levels of glomerular, tubular, oxidative and proinflammatory markers were weekly monitored. RESULTS: Each diabetic group showed a different pattern of inflammatory, oxidative and signs of nephropathy along the study period, but none had a discriminative power until the fourth week. At this time point, levels of urinary transferrin, serum/urinary IL-6 and TNF-α as well as urinary IL-18 were significantly higher in DN group compared to CD (p = 0.0217, <0.0001, 0.0005, 0.0004, 0.0006, 0.0019, respectively). Predictive thresholds of these markers were calculated by receiver operating characteristic (ROC) curve that showed area under curve (AUC) of 0.9375 for transferrin with cut-off value of 35.2 mg/dL and 1.000 for serum/urinary IL-6 and TNF-α and urinary IL-18 with cut-of values 224.1, 82.11, 6.596, 125.9 and 21.86 pg/mL, respectively. CONCLUSION: Urinary transferrin and the inflammatory endpoints proposed in this study might represent promising biomarkers for the early DN onset.

Myrrh mixed with silver nanoparticles demonstrates superior antimicrobial activity against Porphyromonas gingivalis compared to myrrh and silver nanoparticles alone.

INTRODUCTION: Gingivitis is an oral condition characterized by inflammation and bleeding of the gingiva (gums), largely caused by Porphyromonas gingivalis. Oral hygiene options for controlling P. gingivalis include mouthwash containing Commiphora myrrha (myrrh), which has been shown to be effective against the microbe. Silver nanoparticles (SN) have been studied for their antibacterial effect in different oral health applications, including mouthwash. This was an in vitro laboratory study of the anti-microbial actions of myrrh and SN against P. gingivalis. METHODS: We compared the anti-microbial properties against P. gingivalis of four solutions: a) placebo solution, b) myrrh solution (MS), c) MS mixed with silver nanoparticles (MSN), and d) SN suspension alone. Sixteen agar plates were divided into four groups of four plates, and each group was treated with one of the solutions/suspensions. The solution/suspension was administered on the agar disc diffusion method, and inhibition zones (IZs) were measured after 24 (time 1), 48 (time 2), and 72 h (time 3). To characterize MSN and SN, Fourier-transform infrared spectroscopy (FT-IR) was used. UV-Vis spectroscopy and energy dispersive X-ray (EDX) were used to further characterize MSN. RESULTS: After 24 h, the median IZ for the MS plates was 16 mm, and the median IZ for MSN plates was 15 mm. At time 2, the MS median IZ was 15 mm, but the MSN median IZ increased to 18 mm, and the interquartile ranges (IQRs) did not overlap. At time 3, the median IZs was similar again, with MSN and MS having IZs of 16 mm and 15 mm, respectively. SN alone showed no anti-microbial activity. CONCLUSIONS: Our findings show that MSN displayed superior anti-microbial activity against P. gingivalis compared to MS and SN after 48 h of incubation, but not after 24 h. Also, the increased anti-microbial activity had ceased by 72 h.

Circulating macrophage inflammatory protein-1ß/IL-12p40 ratio predicts sofosbuvir-based treatment outcome in HCV- genotype 4 patients.

This study aimed to evaluate the prognostic value of baseline macrophage inflammatory protein (MIP)-1ß/IL12p40 ratio for antiviral treatment outcome in HCV genotype 4 patients. METHODS: Sera of 450 treatment-naïve chronic HCV patients and 50 healthy individuals were collected. Liver transaminases, total bilirubin and albumin were biochemically tested, viral RNA was quantified, and circulating MIP-1ß and IL-12p40 were estimated using human anti-MIP-1ß and IL-12p40 antibodies in Sandwich ELISA. RESULTS: No difference was observed in the baseline chemokines levels between responders and relapsers, but the later had a significantly higher MIP-1ß/IL-12p40 ratio (P< 0.0001). Multivariate regression analysis of baseline characteristics showed that gender, age, viral load, albumin level and chemokine ratios can significantly predict treatment outcome (P= 0.0114, 0.0095, 0.042, 0.0004 and < 0.0001; respectively). Accordingly, a predictive threshold of baseline chemokine ratio was calculated and it showed an AUC of 0.6917 (P= 0.0108; 95% CI: 0.5566 to 0.8268). The calculated threshold for predicting virologic response was 8.245, with positive and negative predictive values of 92.98% and 100%; respectively. The chemokine ratios had significant correlations with liver transaminases in treated groups whether pre or post-treatment. CONCLUSION: Baseline MIP-1ß/IL-12p40 ratio represents a non-invasive prognostic biomarker that would provide shorter treatment duration and minimizes the emergence of drug-resistant variants in HCV genotype 4-patients.

Investigating Helicobacter pylori-related pyloric hypomotility: functional, histological, and molecular alterations.

Multiple theories have been proposed describing the pathogenic mechanisms of Helicobacter pylori (H. pylori)-associated gastric motility disorders. We assessed ex vivo pyloric activity in H. pylori-infected rats, and tried to explore the associated ghrelin hormone alteration and pyloric fibrogenesis. In addition, miR-1 was assessed in pyloric tissue samples, being recently accused of having a role in smooth muscle dysfunction. Ninety adult male Wistar albino rats were assigned into nine groups: 1) control group, 2) sterile broth (vehicle group), 3) amoxicillin control, 4) omeperazole control, 5) clarithromycin control, 6) triple therapy control, 7) H. pylori- group, 8) H. pylori-clarithromycin group, and 9) H. pylori-triple therapy group. Urease enzyme activity was applied as an indicator of H. pylori infection. Ex vivo pyloric contractility was evaluated. Serum ghrelin was assessed, and histological tissue evaluation was performed. Besides, pyloric muscle miR-1 expression was measured. The immunological epithelial to mesenchymal transition (EMT) markers; transforming growth factor ß (TGFß), α-smooth muscle actin (α-SMA), and E-cadherin-3 were also evaluated. By H. pylori infection, a significant (P < 0.001) reduced pyloric contractility index was recorded. The miR-1 expression was decreased (P < 0.001) in the H. pylori-infected group, associated with reduced serum ghrelin, elevated TGFß, and α-SMA levels and reduced E-cadherin levels. Decreased miR-1 and disturbed molecular pattern were improved by treatment. In conclusion, H. pylori infection was associated with reduced miR-1, epithelial to mesenchymal transition, and pyloric hypomotility. The miR-1 may be a target for further studies to assess its possible involvement in H. pylori-associated pyloric dysfunction, which might help in the management of human H. pylori manifestations and complications.NEW & NOTEWORTHY This work is investigating functional, histopathological, and molecular changes underlying Helicobacter pylori hypomotility and is correlating these with miR-1, whose disturbance is supposed to be involved in smooth muscle dysfunction and cell proliferation according to literature. Epithelial to mesenchymal transition and reduced ghrelin hormone may contribute to H. pylori infection-associated hypomotility. H. pylori infection was associated with reduced pyloric miR-1 expression. Targeting miR-1 could be valuable in the clinical management of pyloric hypofunction.

Association between Periodontitis and Genetic Polymorphisms in Interleukins among Patients with Diabetes Mellitus.

There is a perplexity in the association between interleukin (IL) polymorphisms and periodontitis among patients with and without diabetes mellitus (DM). The aim of the present study was to evaluate indexed data regarding the association between periodontitis and genetic polymorphisms in interleukins among patients with and without DM. The addressed question was "Is there an association between periodontitis and polymorphisms in interleukins among patients with and without DM?" Original studies were included. Indexed databases were searched, and the pattern of the present literature review was customized to summaries' the pertinent information. Eight studies were included and processed for data extraction. Two studies showed that polymorphisms in IL-1B genes aggravate periodontitis in patients with type-2 DM, and two studies showed that IL-1B genes either do not or are less likely to contribute towards the progression of periodontitis in patients with type-2 DM. Two studies reported that IL genes do not show cross-susceptibility with periodontitis and type-2 DM. One study reported that the primary factor that governs the occurrence and progression of periodontitis in patients with and without type-2 DM is poor routine oral hygiene maintenance. Seven studies had a high risk of bias. The role of IL gene polymorphisms in the development and progression of periodontitis in patients with and without DM remains controversial.

Combined effect of hydrogen sulfide and mesenchymal stem cells on mitigating liver fibrosis induced by bile duct ligation: Role of anti-inflammatory, anti-oxidant, anti-apoptotic, and anti-fibrotic biomarkers.

Objectives: Liver fibrosis eventually develops into cirrhosis and hepatic failure, which can only be treated with liver transplantation. We aimed to assess the potential role of hydrogen sulfide (H2S) alone and combined with bone marrow-derived mesenchymal stem cells (BM-MSCs) on hepatic fibrosis induced by bile-duct ligation (BDL) and to compare their effects to silymarin. Materials and Methods: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and alkaline phosphatase (ALP) were investigated in serum. Gene expression levels of CBS (cystathionine ß-synthase), CSE (cystathionine γ-lyase), and alpha-smooth muscle actin (α- SMA) were measured in liver tissues using RT-PCR. Hepatic protein kinase (Akt) was assessed by Western blot assay. Liver oxidative stress markers, malondialdehyde (MDA), and reduced glutathione (GSH) were analyzed by the colorimetric method. Lipocalin-2 (LCN2) and transforming growth factor-ß (TGF-ß) were measured using ELIZA. Liver tissues were examined by H&E and Masson trichome staining for detection of liver necrosis or fibrosis. Caspase 3 expression was evaluated by immunohistochemistry. Results: H2S and BM-MSCs ameliorated liver function and inhibited inflammation and oxidative stress detected by significantly decreased serum ALT, AST, ALP, TB, and hepatic MDA, Akt, TGF-ß, LCN2, and α-SMA expression and significantly increased CBS and CSE gene expression levels. They attenuated hepatic apoptosis evidenced by decreased hepatic caspase expression. Conclusion: Combined treatment with H2S and BM-MSCs could attenuate liver fibrosis induced by BDL through mechanisms such as anti-inflammation, anti-oxidation, anti-apoptosis, anti-fibrosis, and regenerative properties indicating that using H2S and MSCs may represent a promising approach for management of cholestatic liver fibrosis.

The Potential Role of Undercarboxylated Osteocalcin Upregulation in Microvascular Insufficiency in a Rat Model of Diabetic Cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy (DCM) is accompanied by microvascular complications that lead to myocardial dysfunction and heart failure. Most conventional therapies cannot ameliorate the microvascular insufficiency in DCM. In this study, we tested the hypothesis that undercarboxylated osteocalcin (ucOC) may be a new adjuvant therapy against the progression of DCM and its underlying microvascular pathology. MATERIALS AND METHODS: Diabetes was induced in Wistar rats with a high-fat diet combined with streptozotocin injections, and ucOC was upregulated after warfarin administration in the treated group. After 8 weeks, cardiac functions were assessed using a Langendorff apparatus. Cardiac tissue samples were also extracted to assess the ucOC receptor and vascular endothelial growth factor (VEGF) for histopathological studies. RESULTS: Both the systolic and the diastolic dysfunction observed in the DCM group were significantly improved after the increase in ucOC blood levels. Significant improvement in VEGF and CD31 expression after warfarin injection was associated with increased capillary density, neovascularization, and decreased myocardial fibrosis together with the reestablishment of myocardial structural and ultrastructural patterns. CONCLUSION: Undercarboxylated osteocalcin may have a promising effect in improving microvascular insufficiency and myocardial dysfunction in DCM.

Effect of gamma-carboxylase inhibition on serum osteocalcin may be partially protective against developing diabetic cardiomyopathy in type 2 diabetic rats.

AIMS: To investigate the possible protective effect of elevated undercarboxylated osteocalcin on diabetic cardiomyopathy mechanisms and risk factors. METHODS: In all, 32 male rats were divided into four groups: control, diabetic, diabetic warfarin and normal warfarin-treated groups. Isolated heart functions were assessed; fasting serum insulin, glucose and glycosylated haemoglobin, homeostasis model assessment insulin resistance and lipid profile were investigated. Serum undercarboxylated osteocalcin and adiponectin were also measured. In cardiac tissue, malondialdehyde content, acyl-CoA dehydrogenase gene expression, Bax/Bcl2 ratio, sarcoendoplasmic reticulum calcium ATPase and osteocalcin receptor (G protein-coupled receptor family C group 6 member A) genes expression were investigated. RESULTS: Prophylactic elevation of undercarboxylated osteocalcin was accompanied by improved insulin sensitivity and lipid profile, increased serum adiponectin, upregulated myocardial osteocalcin receptor with preserved left ventricular function, decreased cardiac malondialdehyde content, acyl-CoA dehydrogenase and Bax/Bcl2 ratio. CONCLUSION: Undercarboxylated osteocalcin was suggested to have protective effects against diabetic cardiomyopathy, possibly through direct action on upregulated G protein-coupled receptor family C group 6 member A and indirectly via adiponectin. These effects may be mediated through antagonizing oxidative stress and apoptosis.

Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate.

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes.

Signaling mechanisms of a water soluble curcumin derivative in experimental type 1 diabetes with cardiomyopathy.

BACKGROUND: Curcumin exhibits anti-diabetic activities, induces heme-oxygenase-1 (HO-1) and is an inhibitor of transcriptional co-activator p300. A novel water soluble curcumin derivative (NCD) has been developed to overcome low invivo bioavailability of curcumin. We evaluated the effect of the NCD on signaling mechanisms involved in cardiomyocyte hypertrophy and studied whether its action is mediated via inducible HO-1. MATERIALS AND METHODS: Rats were divided into controls, controls receiving NCD, diabetic, diabetic receiving NCD, diabetic receiving pure curcumin, diabetic receiving HO inhibitor, zinc protoporphyrin IX (ZnPP IX) and diabetic receiving NCD and ZnPP IX. NCD and curcumin were given orally. After 45 days, cardiac physiologic parameters, plasma glucose, insulin, glycated hemoglobin (GHb), HO-1 gene expression and HO activity in pancreas and cardiac tissues were assessed. Gene expression of p300, atrial natriuretic peptide (ANP) and myocyte enhancer factor 2 (MEF2A and MEF2C) were studied. RESULTS: NCD and curcumin decreased plasma glucose, GHb and increased insulin levels significantly in diabetic rats. This action may be partially mediated by induction of HO-1 gene. HO-1 gene expression and HO activity were significantly increased in diabetic heart and pancreas. Diabetes upregulated the expression of ANP, MEF2A, MEF2C and p300. NCD and curcumin prevented diabetes-induced upregulation of these parameters and improved left ventricular function. The effect of the NCD was better than the same dose of curcumin.

Effect of recombinant erythropoietin on ischemia-reperfusion-induced apoptosis in rat liver

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Ischemia-reperfusion (I/R) cannot be avoided in liver transplantation procedures, and apoptosis is a central mechanism of cell death after liver reperfusion. Protective effect of recombinant erythropoietin (rhEPO) on liver apoptosis has not been clearly investigated. This work investigated intraportal (IP) rhEPO-protective effect in a rat model of hepatic I/R-induced apoptosis and its appropriated time and dose of administration. Eight groups were included (n = 10/group): sham-operated, I/R (45 min ischemia and 2 h reperfusion), preconditioned rhEPO I/R (24 h or 30 min before ischemia), and postconditioned rhEPO I/R (before reperfusion) using two different rhEPO (..) (AU)