Search results for: subcutaneous insulin injection
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 1290

Search results for: subcutaneous insulin injection

1200 Molding Properties of Cobalt-Chrome-Based Feedstocks Used in Low-Pressure Powder Injection Molding

Authors: Ehsan Gholami, Vincent Demers

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Low-pressure powder injection molding is an emerging technology for cost-effectively producing complex shape metallic parts with the proper dimensional tolerances, either in high or in low production volumes. In this study, the molding properties of cobalt-chrome-based feedstocks were evaluated for use in a low-pressure powder injection molding process. The rheological properties of feedstock formulations were obtained by mixing metallic powder with a proprietary wax-based binder system. Rheological parameters such as reference viscosity, shear rate sensitivity index, and activation energy for viscous flow, were extracted from the viscosity profiles and introduced into the Weir model to calculate the moldability index. Feedstocks were experimentally injected into a spiral mold cavity to validate the injection performance calculated with the model.

Keywords: binder, feedstock, moldability, powder injection molding, viscosity

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1199 Concentrations of Leptin, C-Peptide and Insulin in Cord Blood as Fetal Origins of Insulin Resistance and Their Effect on the Birth Weight of the Newborn

Authors: R. P. Hewawasam, M. H. A. D. de Silva, M. A. G. Iresha

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Obesity is associated with an increased risk of developing insulin resistance. Insulin resistance often progresses to type-2 diabetes mellitus and is linked to a wide variety of other pathophysiological features including hypertension, hyperlipidemia, atherosclerosis (metabolic syndrome) and polycystic ovarian syndrome. Macrosomia is common in infants born to not only women with gestational diabetes mellitus but also non-diabetic obese women. During the past two decades, obesity in children and adolescents has risen significantly in Asian populations including Sri Lanka. There is increasing evidence to believe that infants who are born large for gestational age (LGA) are more likely to be obese in childhood. It is also established from previous studies that Asian populations have higher percentage body fat at a lower body mass index compared to Caucasians. High leptin levels in cord blood have been reported to correlate with fetal adiposity at birth. Previous studies have also shown that cord blood C-peptide and insulin levels are significantly and positively correlated with birth weight. Therefore, the objective of this preliminary study was to determine the relationship between parameters of fetal insulin resistance such as leptin, C-peptide and insulin and the birth weight of the newborn in a study population in Southern Sri Lanka. Umbilical cord blood was collected from 90 newborns and the concentration of insulin, leptin, and C-peptide were measured by ELISA technique. Birth weight, length, occipital frontal, chest, hip and calf circumferences of newborns were measured and characteristics of the mother such as age, height, weight before pregnancy and weight gain were collected. The relationship between insulin, leptin, C-peptide, and anthropometrics were assessed by Pearson’s correlation while the Mann-Whitney U test was used to assess the differences in cord blood leptin, C-peptide, and insulin levels between groups. A significant difference (p < 0.001) was observed between the insulin levels of infants born LGA (18.73 ± 0.64 µlU/ml) and AGA (13.08 ± 0.43 µlU/ml). Consistently, A significant increase in concentration (p < 0.001) was observed in C-peptide levels of infants born LGA (9.32 ± 0.77 ng/ml) compared to AGA (5.44 ± 0.19 ng/ml). Cord blood leptin concentration of LGA infants (12.67 ng/mL ± 1.62) was significantly higher (p < 0.001) compared to the AGA infants (7.10 ng/mL ± 0.97). Significant positive correlations (p < 0.05) were observed among cord leptin levels and the birth weight, pre-pregnancy maternal weight and BMI between the infants of AGA and LGA. Consistently, a significant positive correlation (p < 0.05) was observed between the birth weight and the C peptide concentration. Significantly high concentrations of leptin, C-peptide and insulin levels in the cord blood of LGA infants suggest that they may be involved in regulating fetal growth. Although previous studies suggest comparatively high levels of body fat in the Asian population, values obtained in this study are not significantly different from values previously reported from Caucasian populations. According to this preliminary study, maternal pre-pregnancy BMI and weight may contribute as significant indicators of cord blood parameters of insulin resistance and possibly the birth weight of the newborn.

Keywords: large for gestational age, leptin, C-peptide, insulin

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1198 Radial Fuel Injection Computational Fluid Dynamics Model for a Compression Ignition Two-Stroke Opposed Piston Engine

Authors: Tytus Tulwin, Rafal Sochaczewski, Ksenia Siadkowska

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Designing a new engine requires a large number of different cases to be considered. Especially different injector parameters and combustion chamber geometries. This is essential when developing an engine with unconventional build – compression ignition, two-stroke operating with direct side injection. Computational Fluid Dynamics modelling allows to test those different conditions and seek for the best conditions with correct combustion. This research presents the combustion results for different injector and combustion chamber cases. The shape of combustion chamber is different than for conventional engines as it requires side injection. This completely changes the optimal shape for the given condition compared to standard automotive heart shaped combustion chamber. Because the injection is not symmetrical there is a strong influence of cylinder swirl and piston motion on the injected fuel stream. The results present the fuel injection phenomena allowing to predict the right injection parameters for a maximum combustion efficiency and minimum piston heat loads. Acknowledgement: This work has been realized in the cooperation with The Construction Office of WSK "PZL-KALISZ" S.A." and is part of Grant Agreement No. POIR.01.02.00-00-0002/15 financed by the Polish National Centre for Research and Development.

Keywords: CFD, combustion, injection, opposed piston

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1197 Deep Injection Wells for Flood Prevention and Groundwater Management

Authors: Mohammad R. Jafari, Francois G. Bernardeau

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With its arid climate, Qatar experiences low annual rainfall, intense storms, and high evaporation rates. However, the fast-paced rate of infrastructure development in the capital city of Doha has led to recurring instances of surface water flooding as well as rising groundwater levels. Public Work Authority (PWA/ASHGHAL) has implemented an approach to collect and discharge the flood water into a) positive gravity systems; b) Emergency Flooding Area (EFA) – Evaporation, Infiltration or Storage off-site using tankers; and c) Discharge to deep injection wells. As part of the flood prevention scheme, 21 deep injection wells have been constructed to discharge the collected surface and groundwater table in Doha city. These injection wells function as an alternative in localities that do not possess either positive gravity systems or downstream networks that can accommodate additional loads. These injection wells are 400-m deep and are constructed in a complex karstic subsurface condition with large cavities. The injection well system will discharge collected groundwater and storm surface runoff into the permeable Umm Er Radhuma Formation, which is an aquifer present throughout the Persian Gulf Region. The Umm Er Radhuma formation contains saline water that is not being used for water supply. The injection zone is separated by an impervious gypsum formation which acts as a barrier between upper and lower aquifer. State of the art drilling, grouting, and geophysical techniques have been implemented in construction of the wells to assure that the shallow aquifer would not be contaminated and impacted by injected water. Injection and pumping tests were performed to evaluate injection well functionality (injectability). The results of these tests indicated that majority of the wells can accept injection rate of 200 to 300 m3 /h (56 to 83 l/s) under gravity with average value of 250 m3 /h (70 l/s) compared to design value of 50 l/s. This paper presents design and construction process and issues associated with these injection wells, performing injection/pumping tests to determine capacity and effectiveness of the injection wells, the detailed design of collection system and conveying system into the injection wells, and the operation and maintenance process. This system is completed now and is under operation, and therefore, construction of injection wells is an effective option for flood control.

Keywords: deep injection well, flood prevention scheme, geophysical tests, pumping and injection tests, wellhead assembly

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1196 Uniqueness and Repeatability Analysis for Slim Tube Determined Minimum Miscibility Pressure

Authors: Waqar Ahmad Butt, Gholamreza Vakili Nezhaad, Ali Soud Al Bemani, Yahya Al Wahaibi

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Miscible gas injection processes as secondary recovery methods can be applied to a huge number of mature reservoirs to improve the trapped oil displacement. Successful miscible gas injection processes require an accurate estimation of the minimum miscibility pressure (MMP) to make injection process feasible, economical, and effective. There are several methods of MMP determination like slim tube approach, vanishing interfacial tension and rising bubble apparatus but slim tube is the deployed experimental technique in this study. Slim tube method is assumed to be non-standardized for MMP determination with respect to both operating procedure and design. Therefore, 25 slim tube runs were being conducted with three different coil lengths (12, 18 and 24 m) of constant diameter using three different injection rates (0.08, 0.1 and 0.15 cc/min) to evaluate uniqueness and repeatability of determined MMP. A trend of decrease in MMP with increase in coil length was found. No unique trend was found between MMP and injection rate. Lowest MMP and highest recovery were observed with highest coil length and lowest injection rate. It shows that slim tube measured MMP does not depend solely on interacting fluids characteristics but also affected by used coil selection and injection rate choice. Therefore, both slim tube design and procedure need to be standardized. It is recommended to use lowest possible injection rate and estimated coil length depending upon the distance between injections and producing wells for accurate and reliable MMP determination.

Keywords: coil length, injection rate, minimum miscibility pressure, multiple contacts miscibility

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1195 Design and Finite Element Analysis of Clamp Cylinder for Capacity Augmentation of Injection Moulding Machine

Authors: Vimal Jasoliya, Purnank Bhatt, Mit Shah

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The Injection Moulding is one of the principle methods of conversions of plastics into various end products using a very wide range of plastics materials from commodity plastics to specialty engineering plastics. Injection Moulding Machines are rated as per the tonnage force applied. The work present includes Design & Finite Element Analysis of a structure component of injection moulding machine i.e. clamp cylinder. The work of the project is to upgrade the 1300T clamp cylinder to 1500T clamp cylinder for injection moulding machine. The design of existing clamp cylinder of 1300T is checked. Finite Element analysis is carried out for 1300T clamp cylinder in ANSYS Workbench, and the stress values are compared with acceptance criteria and theoretical calculation. The relation between the clamp cylinder diameter and the tonnage capacity has been derived and verified for 1300T clamp cylinder. The same correlation is used to find out the thickness for 1500T clamp cylinder. The detailed design of 1500T cylinder is carried out based on calculated thickness.

Keywords: clamp cylinder, fatigue analysis, finite element analysis, injection moulding machines

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1194 Thermodynamic Modeling of Methane Injection in Gas-Condensate Reservoir Core: A Case Study

Authors: F. S. Alavi, D. Mowla, F. Esmaeilzadeh

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In this paper, the core of Sarkhoon Gas Condensate Reservoir located in the south of Iran was thermodynamically modeled in order to study the natural depletion process and methane injection phenomena for enhanced gas-condensate recovery using the Eclipse 300 compositional simulator. Modeling was performed for three different core lengths with different production and injection flow rates in both vertical and horizontal cases. According to the results, the final condensate in place value in the natural depletion process is approximately independent of the production rate for a given pressure drop. The final condensate in place value is lower in vertical cases compared to horizontal cases. An increase in the injection flow rate leads to a decrease in the percentage of gascondensate recovery. In cores of equal length, gas condensate recovery percent is higher in vertical cases in comparison to horizontal cases. For a constant injection rate, decreasing the core length leads to a decrease in gas condensate recovery.

Keywords: reservoir simulation, methane injection, enhanced condensate recovery, reservoir core, modeling

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1193 Synergistic Effect of Platelet-Rich Plasma with Hyaluronic Acid Injection Following Arthrocentesis to Reduce Pain and Improve Function in Temporomandibular joint (TMJ) Osteoarthritis

Authors: Ayman Hegab

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Increasing evidence supports the use of platelet-rich plasma (PRP) combined with hyaluronic acid (HA) for the treatment of knee osteoarthritis, which effectively promotes cartilage repair. This study aimed to determine whether injection of PRP+HA following arthrocentesis reduces pain and improves maximum incisal opening. This was a single-blind, prospective, randomized control study. The patients were selected based on the Hegab classification: Group I: patients treated with arthrocentesis followed by a single PRP injection; Group II (Control): patients treated with arthrocentesis followed by a single HA injection; and Group III: patients treated with arthrocentesis followed by a single PRP+HA combination injection. The primary predictor variable was the medication used for injection. The primary outcome variables were the maximum voluntary mouth opening and pain index scores. The secondary outcome variable was joint sounds. All outcome variables were assessed and compared among the three groups at baseline and at 1-, 3-, 6-, and 12-month intervals. Other variables, including patients’ age and sex, were evaluated in relation to the patient outcomes. Injecting PRP+HA showed statistically significant improvement in the primary and secondary treatment outcomes over PRP or HA injection throughout the study period (P<0.005). Injection of PRP+HA following arthrocentesis had significant long-term clinical efficacy regarding pain relief that was considered the main concern of both the patient and clinician.

Keywords: TMJ, HA, PRP, osteoarthritis

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1192 Differential Proteomics Expression in Purple Rice Supplemented Type 2 Diabetic Rats’ Skeletal Muscle

Authors: Ei Ei Hlaing, Narissara Lailerd, Sittiruk Roytrakul, Pichapat Piamrojanaphat

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Type 2 diabetes is one of the most common metabolic diseases all over the world. The pathogenesis of type 2 diabetes is not the only dysfunction of pancreatic beta cells but also insulin resistance in muscle, liver and adipose tissue. High levels of circulating free fatty acids, an increased lipid content of muscle cells, impaired insulin-mediated glucose uptake and diminished mitochondrial functioning are pathophysiological hallmarks of diabetic skeletal muscles. Purple rice (Oryza sativa L. indica) has been shown to have antidiabetic effects. However, the underlying mechanism(s) of antidiabetic activity of purple rice is still unraveled. In this research, to explore in-depth cellular mechanism(s), proteomic profile of purple rice supplemented type 2 diabetic rats’ skeletal muscle were analyzed contract with non-supplemented rats. Diabetic rats were induced high-fat diet combined with streptozotocin injection. By using one- dimensional gel electrophoresis (1-DE) and LC-MS/MS quantitative proteomic method, we analyzed proteomic profiles in skeletal muscle of normal rats, normal rats with purple rice supplementation, type 2 diabetic rats, and type 2 diabetic rats with purple rice supplementation. Total 2676 polypeptide expressions were identified. Among them, 24 peptides were only expressed in type 2 diabetic rats, and 24 peptides were unique peptides in type 2 diabetic rats with purple rice supplementation. Acetyl CoA carboxylase 1 (ACACA) found as unique protein in type 2 diabetic rats which is the major enzyme in lipid synthesis and metabolism. Interestingly, DNA damage response protein, heterogeneous nuclear ribonucleoprotein K [Mus musculus] (Hnrnpk), was upregulated in type 2 diabetic rats’ skeletal muscle. Meanwhile, unique proteins of type 2 diabetic rats with purple rice supplementation (bone morphogenetic 7 protein preproprotein, BMP7; and forkhead box protein NX4, Foxn4) involved with muscle cells growth through the regulation of TGF-β/Smad signaling network. Moreover, BMP7 may effect on insulin signaling through the downstream signaling of protein kinase B (Akt) which acts in protein synthesis, glucose uptake, and glycogen synthesis. In conclusion, our study supports that type 2 diabetes impairs muscular lipid metabolism. In addition, purple rice might recover the muscle cells growth and insulin signaling.

Keywords: proteomics, purple rice bran, skeletal muscle, type 2 diabetic rats

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1191 Efficacy of Insulin Pump Therapy on Diabetes Treatment Satisfaction and Glycemic Control among Patients with Type 1 Diabetes Mellitus in Saudi Arabia: A Prospective Study

Authors: Ayman A. Al Hayek, Asirvatham A. Robert, Mohamed A. Al Dawish, Rim B. Braham, Hanouf S. Goudeh, Fahad S. Al Sabaan

Abstract:

Introduction: The aim of this study was to explore the impact of insulin pump therapy on diabetes treatment satisfaction and glycemic control among patients with type 1 diabetes mellitus (T1DM) in Saudi Arabia. Methods: A 6-month, prospective study was conducted among 47 patients (aged17–24 years) with T1DM who attended the Insulin Pump Clinic at Prince Sultan Military Medical City, Riyadh, Saudi Arabia, between April 2014 and November 2014. The respondents were purposively and conveniently selected and were interviewed using the Arabic version of the Diabetes Treatment Satisfaction Questionnaire at baseline, 3, and 6 months. Demographics and clinical variables including hemoglobin A1c (HbA1c) were also collected. Results: The mean (±standard deviation) age of the study cohort was 19.1 ± 1.93 years. Seventeen patients were male (36.2%) and 30 were female (63.8%). Compared to baseline, significant positive differences were found in treatment satisfaction among female patients and patients with long-standing T1DM at 6 months. Frequency of hyperglycemia and hypoglycemia declined significantly in female patient’s at 6 months and in patients who had a shorter duration of T1DM. Furthermore, significant positive differences were found in HbA1c levels among female patients and among those who had a shorter duration of T1DM compared to baseline. Both female and male patients and those with a shorter duration of T1DM showed significant decline in insulin necessity at6months when compared to baseline. Conclusion: Although multiple daily injections is a feasible preference for insulin supply, insulin pumps should also be considered for patients with T1DM as it appears to increase patients’ treatment satisfaction, decrease the frequency of hypoglycemia, hyperglycemia, and reduce HbA1c levels.

Keywords: type 1 diabetes, insulin pump, Saudi Arabia, T1DM

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1190 Synergistic Effect of Curcumin and Insulin on GLUT4 Translocation in C2C12 Cell

Authors: Javad Mohiti-Ardekani, Shabodin Asadii, Ali Moradi

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Introduction: Curcumin, the yellow pigment in turmeric, has been shown as an anti-diabetic agent for centuries but only in recent few years, its mechanism of action has been under investigation. Some studies showed that curcumin might exert its anti-diabetic effect via increasing glucose transporter isotype-4 (GLUT4) gene and glycoprotein contents in cells. To investigate this possibility, we investigate the effects of extract and commercial curcumin with and without insulin on GLUT4 translocation from intracellular compartments of nuclear or endoplasmic reticulum membranes (N/ER) into the cytoplasmic membrane (CM). Methods and Material: C2C12 myoblastic cell line were seeded in DMEM plus 20 % FBS and differentiated to myotubes using 2 % horse serum. After myotubes formation, 40 µmolar Extract and Commercial curcumin, with or without insulin as intervention, and as control 1 % DMSO were added for 3 h. Cells were washed and homogenized followed by ultracentrifuge fractionation, protein separation by SDS-PAGE and GLUT4 detection using semi-quantitative Western blotting. Data analysis was done by two independent samples t-test for comparison of mean ± SD of GLUT4 percent in categories. GLUT4 contents were higher in CM groups curcumin and curcumin with insulin in comparison to 1 % DMSO-treated myotubes control group. Results: As our results have shown extract and commercial curcumin induces GLUT4 translocation from intra-cell into cell surface. The results have also shown synergic effect of curcumin on translocation of GLUT4 from intra-cell into cell surface in the presence of 100 nm insulin. Discussion: We conclude that curcumin may be a choice of type-2 diabetes mellitus treatment because its extract and commercial enhances GLUT4 contents in CM where it facilitates glucose entrance into the cell. However, it is necessary to trace the signaling pathways which are activated by curcumin.

Keywords: Curcumin, insulin, Diabetes type-2, GLUT4

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1189 Effects of Exercise in the Cold on Glycolipid Metabolism and Insulin Sensitivity in Obese Rats

Authors: Chaoge Wang, Xiquan Weng, Yan Meng, Wentao Lin

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Objective: Cold exposure and exercise serve as two physiological stimuli to glycolipid metabolism and insulin sensitivity. So far, it remains to be elucidated whether exercise plus cold exposure can produce an addictive effect on promoting glycolipid metabolism and insulin sensitivity. Methods: 64 SD rats were subjected to high-fat and high-sugar diets for 9-week and sucessfully to establish an obesity model. They were randomly divided into 8 groups: normal control group (NC), normal exercise group (NE), continuous cold control group (CC), continuous cold exercise group (CE), acute clod control group (AC), acute cold exercise group (AE), intermittent cold control group (IC) and intermittent cold exercise group (IE). For continuous cold exposure, the rats stayed in a cold environment all day; for acute cold exposure, the rats were exposed to cold for only 4h before the end of the experiment; for intermittent cold exposure, the rats were exposed to cold for 4h per day. The protocol for treadmill runnings were as follows: 25m/min (speed), 0°C (slope), 30 mins each time, an interval for 10 mins between two runnings, twice/two days, lasting for 5 weeks. Sampling were conducted on the 5th weekend. Blood lipids, free fatty acids, blood glucose (FBG), and serum insulin (FINS) were examined, and the insulin resistance index (HOMA-IR = FBG (mmol/L)×FINS(mIU/L)/22.5) was calculated. SPSS 22.0 was used for statistical analysis of the experimental results, and the ANOVA analysis was performed between groups (p < 0.05 was significant). Results: (1) Compared with the NC group, the FBG of the rats was significantly declined in the NE, CE, AC, AE, and IE groups (p < 0.05), the FINS of the rats was significantly declined in the AE group (p < 0.05), the HOMA-IR of the rats was significantly declined in the NE, CE, AC, AE and IE groups (p < 0.05). Compared with the NE group, the FBG of the rats was significantly declined in the CE, AE, and IE groups (p < 0.05), the FINS and HOMA-IR of the rats were significantly declined in the AE group (p < 0.05). (2) Compared with the NC group, the CHO, TG, LDL-C, and FFA of the rats were significantly declined in CE and IE groups (p < 0.05), the HDL-C of the rats was significantly higher in NE, CC, CE, AE, and IE groups (p < 0.05). Compared with the NE group, the HDL-C of the rats was significantly higher in the CE and IE groups (p < 0.05). Conclusions: Sedentariness or exercise in the acute cold doesn't make sense in the treatment of type 2 diabetes, which led to one-off increases of the body's insulin sensitivity. Exercise in the continuous and intermittent cold can effectively decline the FBG, TC, TG, LDL-C, and FFA levels and increase the HDL-C level and insulin sensitivity in obese rats. These results can impact the prevention and treatment of type 2 diabetes.

Keywords: cold, exercise, insulin sensitivity, obesity

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1188 The Effect of Education on Nurses' Knowledge Level for Ventrogluteal Site Injection: Pilot Study

Authors: Emel Bayraktar, Gulengun Turk

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Introduction and Objective: Safe administration of medicines is one of the main responsibilities of nurses. Intramuscular drug administration is among the most common methods used by nurses among all drug applications. This study was carried out in order to determine determine the effect of education given on injection in ventrogluteal area on the level of knowledge of nurses on this subject. Methods: The sample of the study consisted of 20 nurses who agreed to participate in the study between 01 October and 31 December 2019. The research is a pretest-posttest comparative, quasi-experimental type pilot study. The nurses were given a 4-hour training prepared on injection into the ventrogluteal area. The training consisted of two hours of theoretical and two hours of laboratory practice. Before the training and 4 weeks after the training, a questionnaire form containing questions about their knowledge and practices regarding the injection of the ventrogluteal region was applied to the nurses. Results: The average age of the nurses is 26.55 ± 7.60, 35% (n = 7) of them are undergraduate and 30% (n = 6) of them work in intensive care units. Before the training, 35% (n = 7) of the nurses stated that the most frequently used intramuscular injection site was the ventrogluteal area, and 75% (n = 15) stated that the safest area was the rectus femoris muscle. After the training, 55% (n = 11) of the nurses stated that they most frequently used the ventrogluteal area and 100% (n = 20) of them stated that the ventrogluteal area was the safest area. The average score the nurses got from the premises before the training is 14.15 ± 6.63 (min = 0, max = 20), the total score is 184. The average score obtained after the training was determined as 18.69 ± 2.35 (min = 12, max = 20), and the total score was 243. Conclusion: As a result of the research, it was determined that the training given on the injection of ventrogluteal area increased the knowledge level of the nurses. It is recommended to organize in-service trainings for all nurses on the injection of ventrogluteal area.

Keywords: safe injection, knowledge level, nurse, intramuscular injection, ventrogluteal area

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1187 Effect of 10 Weeks of Aerobic Exercise Training on Serum Concentrations of Surfactant Protein D and Insulin Resistance in Women with Type 2 Diabetes

Authors: Sajjad Rezaei, Mahdieh Molanouri Shamsi, Azadeh Jamali

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Background and purpose: Surfactant protein D (SP-D) is a lung-specific protein that is detectable in human plasma. Effect of exercise training on SP-D levels as well as its relation to metabolic indices is not known. The present study then aimed to investigate the effects of 10 weeks of aerobic training on serum levels of SP-D and insulin resistance in women with type 2 diabetes. Materials and methods: Twenty-two overweight women with type 2 diabetes mellitus were recruited through deliberate sampling and randomly assigned to intervention and control groups (11 in each group). The intervention group underwent a progressive aerobic training program for 10 weeks, 3 days per week, 30-55 min/day at 50-75% heart rate reserve (HRR). Control group continued with its everyday routine. Blood samples were obtained before and after training for biochemical analysis. Within-group and between-group differences were analyzed with paired and independent t-tests in spss software, respectively, and the relation between variables was analyzed with Pearson’s correlation coefficient (all at P = 0.05). Results: Significant differences were observed between groups in leptin, glucose, waist circumference and VO2 max after training. SP-D was decreased and VO2 max was increased significantly in intervention group. However, no significant correlation was observed between SP-D and other variables. Conclusion: Since there was no corresponding decrease in insulin resistance with decreased levels of SP-D, it seems unlikely for SP-D to mediate the association between obesity and insulin resistance in type 2 diabetics.

Keywords: exercise training, SP-D, insulin resistance, type 2 diabetes

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1186 Impact of an Eight-Week High-Intensity Interval Training with Sodium Nitrite Supplementation on TNF-α, MURF1, and PI3K in Type 2 Diabetic Rats

Authors: Samane Eftekhari Ranjbar

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Diabetes mellitus, a metabolic disorder characterized by elevated blood glucose levels, ranks among the leading causes of adult mortality. This study investigates the impact of an eight-week high-intensity interval training (HIIT) program combined with sodium nitrite supplementation on TNF- α, MURF1, and PI3K in a type 2 diabetes rodent model. Elevated TNF-α levels have been associated with insulin resistance, while MURF1 and PI3K play roles in muscle atrophy and insulin signaling pathways, respectively. In this experimental study, 15 eight-week-old rats from the Sara Laboratory Center in Tabriz were assigned to one of five groups: healthy control, diabetic control, diabetic with sodium nitrite supplementation, diabetic with eight weeks of intermittent exercise, and diabetic with eight weeks of interval training plus sodium nitrite supplementation. The HIIT protocol was designed to span eight weeks, with five weekly sessions at specified intensities and durations. Sodium nitrite, known for its vasodilatory and cytoprotective properties, was administered via injection. The findings revealed that the HIIT program and sodium nitrite supplementation influenced the examined biomarkers. ANOVA test outcomes indicated statistically significant differences in TNF- α (P=0.001), MURF1 (P=0.001), and PI3K (P=0.001) concentrations among the various groups. The healthy control group exhibited substantially decreased TNF- α, and MURF1 levels, as well as elevated PI3K levels compared to the diabetic control group. The exercise group, in conjunction with sodium nitrite supplementation, demonstrated a significant rise in PI3K levels (P=0.001) and a decline in TNF- α levels (P=0.018) relative to the diabetic control group. These results suggest that the combined intervention may help improve insulin sensitivity and reduce inflammation. However, MURF1 levels, which are related to muscle atrophy, showed no significant difference (P=0.24). In conclusion, in type 2 diabetic rats, an eight-week high-intensity interval training program with sodium nitrite supplementation does not affect MURF1 levels but does influence PI3K and TNF- α levels. This combination may hold potential for improving insulin sensitivity and reducing inflammation in type 2 diabetes patients, warranting further investigation and potential translation to human clinical trials.

Keywords: high-intensity interval training, sodium nitrate supplementation, type 2 diabetes, tumor necrosis factor-alpha, phosphatidylinositol-3-kinase, muscle RING-finger protein-1

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1185 Effect of Synchronization Protocols on Serum Concentrations of Estrogen and Progesterone in Holstein Dairy Heifers

Authors: K. Shafiei, A. Pirestani, G. Ghalamkari, S. Safavipour

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Use of GnRH or its agonists to increase conception rates should be based on an understanding of GnRH-induced biological effects on the reproductive-endocrine system. This effect may occur through GnRH-stimulated LH surge stimulating production of progesterone by corpus luteum.the aim of this study was to compare the effects on reproductive efficiency of a luteolytic dose of a synthetic prostaglandin Cloprostenol Sodium versus ainjectable progesterone and Luliberin- A on Follicle estrogen and progesterone levels.In this study, we used45 head of holstein dairy heifersin the three treatments, with 15 replicates per treatment were performed in random groups. all the heifers before the projects is began in two steps injection 3 mL CloprostenolSodium with an interval of 11 days been synchronized and 10 days later, second injection of prostaglandin was conducted after that we started below protocol:Control group (daily sodium chloride serum injection 1 cc), Group B: Day Zero, intramuscular injection of 15 mg Luliberin- A + every other day injection of 3 cc progesterone + day 7, injection of Cloprostenol Sodium+ day 9, injection of 15 mg Luliberin- A.Group C: similar to Grop B + daily injection of progesterone after that blood samples was collected and centrifuged.plasma were analysed by ELISA.the analysis of this study uses SPSS data software package and compared between the mean and LS Means LSD test at 5% significance level was used.The results of this study shows that maximum of progesterone plasma levels were in the control gruop (P ≥ 0.05).Therefore, daily injection of progesterone inhibit the growth CL. the most estrogen levels in plasma were in Group C (P ≥ 0.05) thus it can be concluded, rise in endogenous estrogen concentrations normally stimulates the preovulatory LH release in heifers.

Keywords: Luliberin- A, Cloprostenol Sodium, estrogen, progesterone, dairy heifers

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1184 In Ovo Injection of N-Carbamylglutamate Improves Growth Performance, Muscle Fiber Development, and Meat Quality in Broiler Chickens

Authors: Wang Yuan-hao, Habtamu Ayalew, Jing Wang, Shugeng Wu, Kai Qiu, Guanghai Qi, Haijun Zhang

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N-carbamylglutamate (NCG) has emerged as a promising candidate for regulating endogenous arginine synthesis, thereby promoting desirable growth, carcass traits, and muscle development in broilers. Thus, this study aimed to investigate the effects of NCG in ovo feeding on the growth performance, growth hormones, and meat quality of Ross 308 breeder broilers. A total of 1680 embryo eggs were equally allocated into three treatment groups: non punctured control (NC), saline-injected control (SC; 100μL/egg), and N-carbamylglutamate injected group (NCG; 2 mg/egg). The treatment solution was injected into the amniotic cavity of the embryo at 17.5 days of incubation (DOI). For the subsequent 42 days of post hatch experimental sampling, a total of 360 broiler chicks with 6 replications per treatment and 15 chicks per replication were used. Chickens in the NCG group showed significantly higher (P<0.05) body weight gain (BWG) and final body weight (FBW) at both 21 and 42 days after hatch (DAH), while feed conversion efficiency (FCE) was significantly improved (P<0.05) at 42 DAH. The weight and percentage of drums at 21 DAH and the weight and percentage of breast muscle at 42 DAH were significantly higher (P<0.05) in the NCG group. In addition, insulin (INS), growth hormone (GH), and testosterone (T) levels were significantly higher (P<0.05) in the NCG groups at 21 and 42 DAH. Furthermore, triiodothyronine (T3) and tetraiodothyronine (T4) levels were significantly higher (P<0.05) in the NCG treatment group. Interestingly, meat color values were also significantly higher (P<0.05) in the NCG group at 24 hrs postmortem. Collectively, these findings show that 2 mg NCG in ovo injection improves the growth performance and meat quality of broilers; even the effects extend into the market age of the chickens.

Keywords: N-carbamylglutamate, broiler, in ovo injection, growth performance, meat quality

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1183 Optical Heterodyning of Injection-Locked Laser Sources: A Novel Technique for Millimeter-Wave Signal Generation

Authors: Subal Kar, Madhuja Ghosh, Soumik Das, Antara Saha

Abstract:

A novel technique has been developed to generate ultra-stable millimeter-wave signal by optical heterodyning of the output from two slave laser (SL) sources injection-locked to the sidebands of a frequency modulated (FM) master laser (ML). Precise thermal tuning of the SL sources is required to lock the particular slave laser frequency to the desired FM sidebands of the ML. The output signals from the injection-locked SL when coherently heterodyned in a fast response photo detector like high electron mobility transistor (HEMT), extremely stable millimeter-wave signal having very narrow line width can be generated. The scheme may also be used to generate ultra-stable sub-millimeter-wave/terahertz signal.

Keywords: FM sideband injection locking, master-slave injection locking, millimetre-wave signal generation, optical heterodyning

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1182 Effects of Opuntia ficus-indica var. Saboten on Glucose Uptake and Insulin Sensitivity in Pancreatic β Cell

Authors: Kang-Hyun Leem, Myung-Gyou Kim, Hye Kyung Kim

Abstract:

The prickly pear cactus (Opuntia ficus-indica) has a global distribution and have been used for medicinal benefits such as artherosclerosis, diabetes, gastritis, and hyperglycemia. However, very little information is currently available for their mechanism. The prikly pear variety Opuntia ficus-indica var. Saboten (OFS) is widely cultivated in Cheju Island, southwestern region of Korea, and used as a functional food. Present study investigated the effects of OFS on pancreatic β-cell function using pancreatic islet β cells (HIT cell). Alpha-glucosidase inhibition, glucose uptake, insulin secretion, insulin sensitivity, and pancreatic β cell proliferation were determined. The inhibitory effect of ethanol extract of OFS stem on α-glucosidase enzyme was measured in a cell free system. Glucose uptake was determined using fluorescent glucose analogue, 2-NBDG. Insulin secretion was measured by ELISA assay. Cell proliferation was measured by MTT assay. Ethanol extracts of OFS dose-dependently inhibited α-glucosidase activity as well as glucose uptake. Insulinotrophic effect of OFS extract was observed at high glucose media in pancreatic β-islet cells. Furthermore, pancreatic β cell regeneration was also observed.These results suggest that OFS mediates the antidiabetic activity mainly via α-glucosidase inhibition, glucose uptake, and improved insulin sensitivity.

Keywords: prickly pear cactus, Opuntia ficus-indica var. Saboten, pancreatic islet HIT cells, α-glucosidase, glucose uptake, insulinotrophic

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1181 Study on the Thermal Mixing of Steam and Coolant in the Hybrid Safety Injection Tank

Authors: Sung Uk Ryu, Byoung Gook Jeon, Sung-Jae Yi, Dong-Jin Euh

Abstract:

In such passive safety injection systems in the nuclear power plant as Core Makeup Tank (CMT) and Hybrid Safety Injection Tank, various thermal-hydraulic phenomena including the direct contact condensation of steam and the thermal stratification of coolant occur. These phenomena are also closely related to the performance of the system. Depending on the condensation rate of the steam injected to the tank, the injection of the coolant and pressure equalizing timings of the tank are decided. The steam injected to the tank from the upper nozzle penetrates the coolant and induces a direct contact condensation. In the present study, the direct contact condensation of steam and the thermal mixing between the steam and coolant were examined by using the Particle Image Velocimetry (PIV) technique. Especially, by altering the size of the nozzle from which the steam is injected, the influence of steam injection velocity on the thermal mixing with coolant and condensation shall be comprehended, while also investigating the influence of condensation on the pressure variation inside the tank. Even though the amounts of steam inserted were the same in three different nozzle size conditions, it was found that the velocity of pressure rise becomes lower as the steam injection area decreases. Also, as the steam injection area increases, the thickness of the zone within which the coolant’s temperature decreases. Thereby, the amount of steam condensed by the direct contact condensation also decreases. The results derived from the present study can be utilized for the detailed design of a passive safety injection system, as well as for modeling the direct contact condensation triggered by the steam jet’s penetration into the coolant.

Keywords: passive safety injection systems, steam penetration, direct contact condensation, particle image velocimetry

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1180 Managing Subretinal Bleeds with Intravitreal Aflibercept

Authors: Prachi Abhishek Dave, Abhishek Dave

Abstract:

Purpose: The purpose of this study is to elucidate the role of intravitreal injection Aflibercept in managing complex cases of Wet Age Related Macular Degeneration (ARMD) and the gratifying visual recovery experienced with a minimally invasive procedure. Methods: A 73-year-old gentleman presented with a drop in vision in the left eye for 25 days. On examination, his best corrected visual acuity (BCVA) in the Right eye (OD) was 6/60, and finger counting close to face in the Left eye (OS). On multimodal imaging, he was diagnosed to have a scarred Wet ARMD in OD and an active Wet ARMD with a large subretinal bleed secondary to Wet ARMD in OS. Treatment management options included monotherapy with an Injection Aflibercept or an intravitreal gas injection with tPA followed by Injection Aflibercept. Considering his one-eyed status, the patient decided to go for Aflibercept monotherapy. Results: After 3 monthly injections of injection Aflibercept, the subretinal bleed reduced, the subretinal fluid resolved, and his vision in OS improved to 6/9. He is on a regular follow-up and has not needed any further injections in OS and he maintains 6/9 vision. Conclusions: Conventional treatment guidelines for a large subretinal bleed dictate the use of gas followed by intravitreal Injection Aflibercept. However, gas has its own limitations of causing a rise in intraocular pressure and a transient loss of vision, which is particularly troublesome in one-eyed patients. Injection Aflibercept offers a much safer, less invasive, and elegant treatment option for such patients with equally good or even better visual outcomes.

Keywords: wet ARMD, subretinal bleed, intravitreal injections, aflibercept, EYELEA, intravitreal gas

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1179 Effect of Wettability Alteration in Low Salt Water Injection Modeling

Authors: H. Vahdani

Abstract:

By the adsorption of polar compounds and/or the deposition of organic material, the wettability of originally water-wet reservoir rock can be altered. The degree of alteration is determined by the interaction of the oil constituents, the mineral surface, and the brine chemistry. Recently improving oil recovery by tuning wettability alteration is believed as a new recovery method. Various researchers have demonstrated that low salt water injection has a significant impact on oil recovery. It has been shown, for instance, that additional oil can be produced from reservoir rock by managing the injection water. Large wettability sensitivity has been observed, indicating that the oil/water capillary pressure profiles play a major role during low saline water injection simulation. Although the exact physics on how this alteration occurs is still a research topic; however, it has been reported that some of its effect can be captured by a relative permeability shift from an oil-wet system to a water-wet system. Modeling of low salt water injection mainly is based on the theory of wettability alteration and is hence strongly dependent on the wettability of the reservoir. In this article, combination of different wettabilities has been simulated and it is observed that the highest recoveries were from the cases were the reservoir initially was water-wet, and the lowest recoveries was from the cases were the reservoir initially was considered oil-wet. However for the cases where the reservoir initially was oil-wet, the effect of low-salinity waterflooding was the largest.

Keywords: low salt water injection, wettability alteration, modelling, relative permeability

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1178 Failure Analysis of the Gasoline Engines Injection System

Authors: Jozef Jurcik, Miroslav Gutten, Milan Sebok, Daniel Korenciak, Jerzy Roj

Abstract:

The paper presents the research results of electronic fuel injection system, which can be used for diagnostics of automotive systems. In the paper is described the construction and operation of a typical fuel injection system and analyzed its electronic part. It has also been proposed method for the detection of the injector malfunction, based on the analysis of differential current or voltage characteristics. In order to detect the fault state, it is needed to use self-learning process, by the use of an appropriate self-learning algorithm.

Keywords: electronic fuel injector, diagnostics, measurement, testing device

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1177 Parameter Estimation of Additive Genetic and Unique Environment (AE) Model on Diabetes Mellitus Type 2 Using Bayesian Method

Authors: Andi Darmawan, Dewi Retno Sari Saputro, Purnami Widyaningsih

Abstract:

Diabetes mellitus (DM) is a chronic disease in human that occurred if pancreas cannot produce enough of insulin hormone or the body uses ineffectively insulin hormone which causes increasing level of glucose in the blood, or it was called hyperglycemia. In Indonesia, DM is a serious disease on health because it can cause blindness, kidney disease, diabetic feet (gangrene), and stroke. The type of DM criteria can also be divided based on the main causes; they are DM type 1, type 2, and gestational. Diabetes type 1 or previously known as insulin-independent diabetes is due to a lack of production of insulin hormone. Diabetes type 2 or previously known as non-insulin dependent diabetes is due to ineffective use of insulin while gestational diabetes is a hyperglycemia that found during pregnancy. The most one type commonly found in patient is DM type 2. The main factors of this disease are genetic (A) and life style (E). Those disease with 2 factors can be constructed with additive genetic and unique environment (AE) model. In this article was discussed parameter estimation of AE model using Bayesian method and the inheritance character simulation on parent-offspring. On the AE model, there are response variable, predictor variables, and parameters were capable of representing the number of population on research. The population can be measured through a taken random sample. The response and predictor variables can be determined by sample while the parameters are unknown, so it was required to estimate the parameters based on the sample. Estimation of AE model parameters was obtained based on a joint posterior distribution. The simulation was conducted to get the value of genetic variance and life style variance. The results of simulation are 0.3600 for genetic variance and 0.0899 for life style variance. Therefore, the variance of genetic factor in DM type 2 is greater than life style.

Keywords: AE model, Bayesian method, diabetes mellitus type 2, genetic, life style

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1176 Plasmablastic Lymphoma a New Entity in Patients with HIV Infections

Authors: Rojith K. Balakrishnan

Abstract:

Plasmablastic lymphoma (PBL) is an uncommon, recently described B-cell derived lymphoma that is most commonly seen in patients with Human Immunodeficiency Virus (HIV) infection. Here we report a case of PBL in a 35 year old man with HIV who presented with multiple subcutaneous swellings all over the body and oral mucosal lesions.The biopsy report was suggestive of Diffuse Large B Cell Lymphoma. Immunohistochemistry was done which showed, lymphoma cells, positive for MUM1, CD 138, and VS 38. The proliferation index (MIB) was 95%. Final report was consistent with the diagnosis of Plasmablastic Lymphoma. The lesion completely regressed after treatment with systemic chemotherapy. Up to date, only a few cases of plasmablastic lymphoma have been reported from India. Increased frequency of this lymphoma in HIV patients and rarity of the tumour, along with rapid response of the same to chemotherapy, make this case a unique one. Hence the knowledge about this new entity is important for clinicians who deal with HIV patients.

Keywords: human immunodeficiency virus (HIV), oral cavity lesion, plasmablastic lymphoma, subcutaneous swelling

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1175 Effect of Maternal Factors and C-Peptide and Insulin Levels in Cord Blood on the Birth Weight of Newborns: A Preliminary Study from Southern Sri Lanka

Authors: M. H. A. D. de Silva, R. P. Hewawasam, M. A. G. Iresha

Abstract:

Macrosomia is common in infants born to not only women diagnosed with gestational diabetes mellitus but also non-diabetic obese women. Maternal Body Mass Index (BMI) correlates with the incidence of large for gestational age infants. Obesity has reached epidemic levels in modern societies. During the past two decades, obesity in children and adolescents has risen significantly in Asian populations including Sri Lanka. There is increasing evidence to believe that infants who are born large for gestational age are more likely to be obese in childhood and adolescence and are at risk of cardiovascular and metabolic complications later in life. It is also established that Asians have lower skeletal muscle mass, low bone mineral content and excess body fat for a given BMI indicating a genetic predisposition in the occurrence of obesity. The objective of this study is to determine the effect of maternal weight, weight gain during pregnancy, c-peptide and insulin concentrations in the cord blood on the birth of appropriate for and large for gestational age infants in a tertiary care center in Southern Sri Lanka. Umbilical cord blood was collected from 90 newborns (Male 40, Female 50; gestational age 35-42 weeks) after double clamping the umbilical cord before separation of the placenta and the concentration of insulin and C-peptide were measured by ELISA technique. Anthropometric parameters of the newborn such as birth weight, length, ponderal index, occipital frontal, chest, hip and calf circumferences were measured, and characteristics of the mother were collected. The relationship between insulin, C-peptide and anthropometrics were assessed by Spearman correlation. The multiple logistic regression analysis examined influences of maternal weight, weight gain during pregnancy, C-peptide and insulin concentrations in cord blood as covariates on the birth of large for gestational age infants. A significant difference (P<0.001) was observed between the insulin levels of infants born large for gestational age (18.73 ± 0.52 µlU/ml) and appropriate for gestational age (13.08 ± 0.56 µlU/ml). Consistently, A significant decrease in concentration (41.68%, P<0.001) was observed between C-peptide levels of infants born large for gestational age and appropriate for gestational age. Cord blood insulin and C-peptide levels had a significant correlation with birth weight (r=0.35, P<0.05) of the newborn at delivery. Maternal weight and BMI which are indicators of maternal nutrition were proven to be directly correlated with birth weight and length. To our knowledge, this relationship was investigated for the first time in a Sri Lankan setting and was also evident in our results. This study confirmed the fact that insulin and C-peptide play a major role in regulating fetal growth. According to the results obtained in this study, we can suggest that the increased BMI of the mother has a direct influence on increased maternal insulin secretion, which may subsequently affect cord insulin and C-peptide levels and also birth weight of the infant.

Keywords: C-peptide, insulin, large for gestational age, maternal weight

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1174 The Effect of Intrathecal Adenosine in Control of Neuropathic Pain after Lumbar Discectomy in One Level

Authors: Dawood Aghamohammadi, Mahmoud Eidi, Alireza Pishgahi, Azam Esmaeilnejad

Abstract:

Adenosine has an analgesic and anti-inflammatory role and its injections are used for peri-operative pain management. We want to study efficacy of intrathecal injection of adenosine for post operative radicular pain after lumbar discectomy. 40 patients with unilevel lumbar discectomy who had radicular lower limb pain were treated by 1000 micrograms of intrathecal injection of adenosine. Pain severity, pain killer consumption per day and sleep quality were assessed during a 3 months follow up period. Radicular pain severity was significantly reduced in 3 month follow-up period in comparison to the baseline (F=19760, DF=2.53, p-value<0.001). Further painkiller medication consumption rate in average during 3 month follow-up period after injection was significantly lower in comparison to baseline (F= 19.244, df= 1.98, p-value<0.001). This study suggests that intrathecal injection of adenosine is a safe method in order to reduce postoperative pain after lumbar discectomy.

Keywords: adenosine, intrathecal injection, discectomy, neuropathic pain

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1173 Quantitative Analysis of Orphan Nuclear Receptors in Insulin Resistant C2C12 Skeletal Muscle Cells

Authors: Masocorro Gawned, Stephen Myers, Guat Siew Chew

Abstract:

Nuclear Receptors (NR) are a super family of transcription factors that play a major role in lipid and glucose metabolism in skeletal muscle. Recently, pharmacological evidence supports the view that stimulation of nuclear receptors alleviates Type 2 Diabetes (T2D). The orphan nuclear receptors (ONR) are members of the nuclear receptor (NR) superfamily whose ligands and physiological functions remain unknown. To date, no systematic studies have been carried out to screen for ONRs expressed in insulin resistant (IR) skeletal muscle cells. Therefore, in this study, we have established a model for IR by treating C2C12 skeletal muscle cells with insulin (10nM) for 48 hours. Western Blot analysis of phosphorylated AKT confirmed IR. Real-time quantitative polymerase chain reaction (qPCR) results highlighted key ONRs including NUR77 (NR4A1), NURR1 (NR4A2) and NOR1 (NR4A3) which have been associated with fatty acid oxidation regulation and glucose homeostasis. Increased mRNA expression levels of estrogen-related receptors (ERRs), REV-ERBα, NUR77, NURR1, NOR1, in insulin resistant C2C12 skeletal muscle cells, indicated that these ONRs could potentially play a pivotal regulatory role of insulin secretion in lipid metabolism. Taken together, this study has successfully contributed to the complete analysis of ONR in IR, and has filled in an important void in the study and treatment of T2D.

Keywords: type 2 diabetes, orphan nuclear receptors, transcription receptors, quantitative mRNA expression

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1172 Numerical Analysis of Engine Performance and Emission of a 2-Stroke Opposed Piston Hydrogen Engine

Authors: Bahamin Bazooyar, Xinyan Wang, Hua Zhao

Abstract:

As a zero-carbon fuel, hydrogen can be used in combustion engines to avoid carbon emissions. This paper numerically investigates the engine performance of a two-stroke opposed piston hydrogen engine by using three-dimensional (3D) Computational Fluid Dynamics (CFD) simulations. The engine displacement is 12.2 cm, and the compression ratio of 39. RANS simulations with the k-ε turbulence model and coupled chemistry combustion models are performed at an engine speed of 4500 rpm and hydrogen flow rate of up to 100 gr/s. In order to model the hydrogen injection process, the hydrogen nozzle was meshed with refined mesh, and injection pressure varied between 100 and 200 bars. In order to optimize the hydrogen combustion process, the injection timing was optimized between 15 before the top dead center and 10. The results showed that the combustion efficiency was mostly influenced by the injection pressures due to its impact on the fuel/air mixing and charge inhomogeneity. Nitrogen oxide (NOₓ) emissions are well correlated with engine peak temperatures, demonstrating that the thermal NO mechanism is dominant under engine conditions. Through the optimization of hydrogen injection timing and pressure, the peak thermal efficiency of 45 and NOx emission of 15 ppm/kWh can be achieved at an injection timing of 350 CA and pressure of 160 bars.

Keywords: engine, hydrogen, diesel, two-stroke, opposed-piston, decarbonisation

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1171 Production of Insulin Analogue SCI-57 by Transient Expression in Nicotiana benthamiana

Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

Abstract:

The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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