From in-silico immunogenicity verification to in vitro expression of recombinant Core-NS3 fusion protein of HCV.

BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is a serious global health burden. There is no effective vaccine against HCV and new direct acting antivirals (DAAs) are so expensive and virtually unavailable to the public. Therefore, seeking for therapeutic or prophylactic vaccines is exigent and reliever. METHODS: The secondary and tertiary structures of the recombinant Core-NS3 (rC-N) fusion protein of HCV and its B and T-cells epitopes were evaluated with bioinformatics software. Cloning and in vitro expression of rC-N were performed by pET24a(+) and E.coli BL21-DE3 expression host, respectively. The recombinant protein purification was done by affinity chromatography method and then identified by Western blotting using anti-His monoclonal antibody. RESULTS: The sequences of rC-N protein consist of 1-118 amino acid parts of Core and 1095-1384 amino acids of NS3 were connected by a flexible linker (AAY) with proteasome cleavable site. The expressed and purified 46.7292 kDa rC-N protein had antigenic value up to threshold and conservancy found in this chimeric protein. Ramchandran Plot analysis represented that most residues were fallen in favourable regions. It also interacted with both type I and II major histocompatibility complex (MHC I, II) molecules. The rC-N had antigenic behaviour to create T cell responses. CONCLUSION: The results indicated that conserved rC-N protein had the ability to induce T-cell-mediated immune responses and it could be utilized as a therapeutic vaccine candidate against HCV (Tab. 3, Fig. 4, Ref. 40).

Evidence of a role of ANGPTL6 in resting metabolic rate and its potential application in treatment of obesity.

AIM: ANGPTL6 (Angiopoietin-related growth factor 6) is a circulating protein which is suggested to antagonize obesity. The purpose of this study was to evaluate a potential relationship between fasting serum ANGPTL6 and resting metabolic rate (RMR) as well as the body composition in obese and subjects with normal weight. METHODS: Participants were 62 obese and 41 non-obese subjects who were assessed following an overnight fasting for RMR by means of indirect calorimetry. Body composition was measured using Bodystat devise. Serum ANGPTL6 levels were quantified by ELISA method. RESULTS: Based on ROC analysis best RMR/kg cut-off value for predicting the risk of obesity was 20 kcal/24h /kg. The participants with RMR/kg≥20 kcal/24h/kg were considered as and subjects with RMR/kg<20 kcal/24h/kg were categorized as. In group I, 72.3% of subjects were obese, whereas, 47.4% subjects in group II were suffering from the disease. Participants in group II who showed significantly lower HDL and ANGPTL6 levels. Moreover, we found significantly higher triglyceride and hs-CRP levels in this group. There was significant difference in weight, body mass index, fat mass, visceral fat, RMR/kg, fasting serum glucose, insulin and hs-CRP among those with different levels of the serum ANGPTL6 concentration. We found higher values of RMR/kg in subjects with higher circulating ANGPTL6 concentration. CONCLUSION: ANGPTL6 affects RMR and significantly improves lipid profile and slightly does so regarding insulin concentrations and sensitivity to it. Further study is warranted as it seems that the results of this study might potentially lead to advent of a pharmacological treatment for obesity.

Lysophosphatidic Acid Alters The Expression of Apoptosis Related Genes and miR-22 in Cultured and Autotransplanted Ovaries.

OBJECTIVE: The aim of this study was to evaluate the effect of lysophosphatidic acid (LPA) on the follicular development, incidence of cell death, and expressions of apoptosis related genes and miR-22 in transplanted ovaries. MATERIALS AND METHODS: In this experimental study, three-week-old mice ovaries were cultured for 24 hours in the presence and absence of LPA, and we assessed cell survival and normal follicular rates in some of the cultured ovaries. The remaining cultured ovaries were autotransplanted in the presence and absence of LPA as four experimental groups (LPA-/LPA-, LPA-/LPA+, LPA+/LPA-, LPA+/LPA+). The follicular development, immunohistochemistry for BAX, and expressions of genes related to apoptosis and miR-22 by real time reverse transcription polymerase chain reaction (RTPCR) were studied at the first oestrous cycles in the recovered ovaries. Sera 17-ß-oestradiol (E2) and progesterone (P4) levels were also assessed. RESULTS: Both cell survival and normal follicular rates were significantly higher in cultured ovaries in the presence of LPA after 24 hours (P<0.05). There was an increase in follicular development in comparison with the intact control group in the four transplanted groups (P<0.05). The LPA+/LPA- group had significantly higher follicular development, a decline in BAX positive cells, and a decrease in pro-apoptotic gene expressions in parallel with enhanced expression of anti-apoptotic and miR-22 genes and higher levels of hormones compared with the non-treated and intact control groups (P<0.05). CONCLUSION: LPA, as a survival factor, improves follicular development in transplanted ovaries by providing a balance between the anti- and pro-apoptotic genes in association with an increase in miR-22 expression.

HLA-DRB1*1501 intensifies the impact of IL-6 promoter polymorphism on the susceptibility to multiple sclerosis in an Iranian population.

BACKGROUND: The multifunctional cytokine interleukin-6 (IL-6) is involved in inflammatory processes in the central nervous system. It is well documented that amount of IL-6 is increased in serum, cerebrospinal fluid and central nervous system lesions of patients with multiple sclerosis. A single nucleotide polymorphism at position -174 in the IL-6 gene promotor appears to influence IL-6 expression. Recently, several researchers have focused on HLA-DRB alleles, specifically HLA-DRB1*1501, as a potential risk allele in the pathogenesis of multiple sclerosis. OBJECTIVE: To investigate the possible influence of IL-6/-174 polymorphisms on susceptibility to multiple sclerosis and its integration with HLA-DRB1*1501. Genomic DNA was extracted from whole blood of 345 patients with multiple sclerosis and 426 control subjects. METHOD: The SSP-PCR method was used to determine genotypes and Fisher's exact test was applied to determine differences between groups. HLA-DRB1*1501 was observed more frequently among multiple sclerosis patients compared with healthy subjects (45% and 34%, respectively; OR = 1.6, 95% CI = 1.2-2.2, p = 0.0018). At the IL-6/-174 position, the G allele had higher frequency among multiple sclerosis patients compared with controls (77% and 70%, respectively; OR = 1.4, 95% CI = 1.1-1.8, p = 0.0038). This difference was more significant among HLA-DRB1*1501-positive patients and controls (81% and 67%, respectively; OR = 1.9, 95% CI = 1.5-2.5, p < 0.0001). RESULTS: Our results have shown that the G allele at the IL-6/-174 promoter polymorphism may be associated with development of multiple sclerosis in this population, and may be strengthened by HLA-DRB1*1501. CONCLUSIONS: We suggest more studies to confirm these results in other populations.

Molecular characterization of Iranian patients with type 3 von Willebrand disease.

von Willebrand's disease (VWD) type 3 is a rare but severe autosomal-recessive inherited bleeding disorder with a prevalence higher in certain locations where consanguineous marriages are relatively frequent. The genetic defects causing recessive type 3 VWD in 10 unrelated families from Iran have been investigated and the genetic heterogeneity among these patients was evaluated. All exons and their flanking regions of von Willebrand factor gene were amplified by PCR and sequenced using specific primers. Eight patients were fully characterized at the molecular level. Six different gene alterations were identified. All the mutations caused null alleles, three being nonsense mutations (Q104X, Q793X and E1981X), two possible splice site mutations (2443-1G>C and 1110-1G>A) and one small deletion (3237delA). Three of them have not been described previously. Most patients were born from consanguineous marriages and all were homozygous for their mutations. The results confirm that molecular defects in type 3 VWD are heterogeneous with mutations arising randomly within the entire gene.

Effect of Ramadan fasting on some indices of insulin resistance and components of the metabolic syndrome in healthy male adults.

The purpose of the present study was to evaluate the effect of Ramadan fasting on insulin sensitivity in subjects with the metabolic syndrome. Males (n 55; age 34.1 (sd 8.9) years) with the metabolic syndrome were studied. Blood pressure, waist circumference, body weight, HDL-cholesterol (HDL-C), TAG, fasting plasma glucose (FPG), fasting blood insulin and insulin resistance indices (quantitative insulin sensitivity check index (QUICKI), homeostasis model assessment of insulin resistance (HOMA-IR) and reciprocal index of HOMA-IR (1/HOMA-IR)) were evaluated before and after 30 d of Ramadan fasting (two meals at 12 h intervals). The dietary intake was estimated by 24 h recall before and after fasting. The total daily energy intake was decreased by 234.6 (sd 88.2) kJ/d in the fasting period (P = 0.005). 1/HOMA-IR, QUICKI and HDL-C were significantly increased (P = 0.005, P = 0.001 and P = 0.004) and FPG significantly decreased (P < 0.005) after fasting. Simple linear regression analysis demonstrated that HOMA-IR, 1/HOMA-IR and QUICKI were related to waist circumference after intervention (r 0.458, P < 0.001; r - 0.396, P < 0.05; r - 0.342, P < 0.05). In conclusion, the present study showed that the combined change in the number and timing of meals and portioning of the entire intake into only two meals per d may increase insulin sensitivity in subjects with the metabolic syndrome even when the decrease in energy consumption is minimal.

Characterization of the interaction between von Willebrand factor and osteoprotegerin.

BACKGROUND AND OBJECTIVE: Osteoprotegerin (OPG), a member of the tumor necrosis-factor receptor superfamily, plays an important role in bone remodeling and is also involved in vascular diseases. OPG is physically associated with von Willebrand factor (VWF), a glycoprotein involved in primary hemostasis, within the Weibel-Palade bodies (WPBs) of endothelial cells and in plasma. The present study aimed to elucidate the molecular mechanisms underlying the interaction between OPG and VWF. METHODS AND RESULTS: In a solid-phase binding assay, VWF was able to bind specifically to OPG in a calcium-dependent manner. This interaction displayed strong pH dependence with optimal binding occurring at pH 6.5 and was severely impaired by chloride-ion concentrations above 40 mm. Using a series of purified VWF derivatives the functional site that supports VWF interaction with OPG was localized on its Al domain. Fluorescence microscopy on human umbilical vein endothelial cells showed co-localization of VWF and OPG in WPBs. When secretion was induced, OPG remained associated with VWF in extracellular patches of release under biochemical conditions found in blood plasma. CONCLUSIONS: Our observations demonstrate the existence of an interactive site for OPG within the VWF A1-domain. This study established that the optimal biochemical parameters allowing a complex formation between VWF and OPG are those thought to prevail in the trans-Golgi network. These conditions would allow VWF to act as a cargo targeting OPG to WPBs. Finally, blood environments appear suitable to preserve the complex, which may participate in vascular injury, arterial calcification and inflammation.

In vitro study of a new biodegradable nanocomposite based on poly propylene fumarate as bone glue.

A novel poly propylene fumarate (PPF)-based glue which is reinforced by nanobioactive glass (NBG) particles and promoted by hydroxyethyl methacrylate (HEMA) as crosslinker agent, was developed and investigated for bone-to-bone bonding applications. In-vitro bioactivity, biodegradability, biocompatibility, and bone adhesion were tested and the results have verified that it can be used as bone glue. In an in-vitro condition, the prepared nanocomposite (PPF/HEMA/NBG) showed improved adhesion to wet bone surfaces. The combined tension and shear resistance between two wet bone surfaces was measured, and its maximum value was 9±59MPa. To investigate the bioactivity and biodegradability of the nanocomposite, it has been immersed in simulated body fluid (SBF). After 14days exposure to SBF, a hydroxyapatite (HA) layer formed on the surface of the composite confirms the bioactivity of this material. In the XRD pattern of the nanocomposite surface, the HA characteristic diffraction peak at θ=26 and 31.8 were observed. Also, by monitoring the weight change after 8weeks immersion in SBF, the mass loss was about 16.46wt%. It has been confirmed that this nanocomposite is a biodegradable material. Also, bioactivity and biodegradability of nanocomposite have been proved by SEM images. It has been showed that by using NBG particles and HEMA precursor, mechanical properties increased significantly. The ultimate tensile strength (UTS) of nanocomposite which contains 20% NBG and the ratio of 70/30wt% PPF/HEMA (PHB.732) was approximately 62MPa, while the UTS in the pure PPF/HEMA was about 32MPa. High cell viability in this nanocomposite (MTT assays, 85-95%) can be attributed to the NBG nature which contains calcium phosphate and is similar to physiological environment. Furthermore, it possesses biomineralization and biodegradation which significantly affected by impregnation of hydrophilic HEMA in the PPF-based polymeric matrix. The results indicated that the new synthesized biodegradable PPF/HEMA/NBG composite is suitable for biomedical applications especially as biodegradable bone glue in orthopedic surgeries.

Liver transplantation and aortic valve replacement.

Surgical procedures involving heart and liver are rare and have been limited to either combined heart and liver transplantation or coronary artery bypass graft surgery (CABG) or aortic valve surgery and orthotopic liver transplantation (OLT). Aortic valve replacement (AVR) and pulmonary valve vegetectomy for bacterial endocarditis after OLT have also been reported. There are only five cases with aortic stenosis and cirrhosis reported to have combined AVR and liver transplantation. In the presence of cirrhosis, AVR has a significant risk for mortality because of bleeding from coagulopathy, renal failure, infection, and poor post-operative wound healing. Herein, we report on a case and management analysis of combined sequential AVR, and OLT in a 40-year-old cirrhotic man with Child and MELD score of C and 29, respectively. Echocardiography detected severe aortic insufficiency (AI) with enlarged left ventricle. Due to severe AI, the cardiologist recommended AVR prior to transplantation. The patient underwent metallic AVR. 4 months later, he received OLT. Both operations were successful and uneventful. Prioritizing AVR before OLT was successful in this patient. However, each patient must be evaluated individually and multiple factors should be assessed in pre-operation evaluation.