Search results for: high protein

Authors: Fei Xu, Guofan Zhang, Xiao Liu

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Many major marine invertebrate phyla are characterized by indirect development. These animals transit from planktonic larvae to benthic adults via settlement and metamorphosis, which has many advantages for organisms to adapt marine environment. Studying the biological process of metamorphosis is thus a key to understand the origin and evolution of indirect development. Although the mechanism of metamorphosis has been largely studied on their relationships with the marine environment, microorganisms, as well as the neurohormones, little is known on the gene regulation network (GRN) during metamorphosis. We treated competent oyster pediveligers with epinephrine, which was known to be able to effectively induce oyster metamorphosis, and analyzed the dynamics of gene and proteins with transcriptomics and proteomics methods. The result indicated significant upregulation of protein synthesis system, as well as some transcription factors including Homeobox, basic helix-loop-helix, and nuclear receptors. The result suggested the GRN complexity of the transition stage during oyster metamorphosis.

Keywords: indirect development, gene regulation network, protein synthesis, transcription factors

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Authors: L. Piñeiro, N. Cobas, L. Gómez-Limia, S. Martínez, I. Franco

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The swordfish (Xiphias gladius) is a large oceanic fish of high commercial value, which is widely distributed in waters of the world’s oceans. They are considered to be an important source of high quality proteins, vitamins and essential fatty acids, although only half of the population follows the recommendation of nutritionists to consume fish at least twice a week. Swordfish is consumed worldwide because of its low fat content and high protein content. It is generally sold as fresh, frozen, and as pieces or slices. The aim of this study was to evaluate the effect of salting and frying on the composition of the water-soluble vitamins (B₂, B₃, B₉ and B₁₂) and fat-soluble vitamins (A, D, and E) of swordfish. Three loins of swordfish from Pacific Ocean were analyzed. All the fishes had a weight between 50 and 70 kg and were transported to the laboratory frozen (-18 ºC). Before the processing, they were defrosted at 4 ºC. Each loin was sliced and salted in brine. After cleaning the slices, they were divided into portions (10×2 cm) and fried in olive oil. The identification and quantification of vitamins were carried out by high-performance liquid chromatography (HPLC), using methanol and 0.010% trifluoroacetic acid as mobile phases at a flow-rate of 0.7 mL min^-1. The UV-Vis detector was used for the detection of the water- and fat-soluble vitamins (A and D), as well as the fluorescence detector for the detection of the vitamin E. During salting, water and fat-soluble vitamin contents remained constant, observing an evident decrease in the values of vitamin B₂. The diffusion of salt into the interior of the pieces and the loss of constitution water that occur during this stage would be related to this significant decrease. In general, after frying water-soluble and fat-soluble vitamins showed a great thermolability with high percentages of retention with values among 50–100%. Vitamin B₃ is the one that exhibited higher percentages of retention with values close to 100%. However, vitamin B₉ presented the highest losses with a percentage of retention of less than 20%.

Keywords: frying, HPLC, salting, swordfish, vitamins

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Authors: Padmanabhan D, Kavitha S

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The purpose of the study is to investigate arsenic resistance ability of indigenous microflora and its ability to utilize arsenic species form containing water source. PS-6 potential arsenic tolerance bacterium was screened from thirty isolates from Pichavaram Mangroves of India having tolerance to grow up to 1000 mg/l of As (V) and 800 mg/l of As (III) and arsenic utilization ability of 98 % of As (V) and 97% of As (III) with initial concentration of 3-5 mg/l within 48 hrs. Optimum pH and temperature was found to be ~7-7.4 and 37°C. Active growth of PS-6 in minimal salt media (MSB) helps in cost effective biomass production. Dry weight analysis of PS-6 has shown significant difference in biomass when exposed to As (III) and As (V). Protein level study of PS-6 after exposing to As (V) and As (III) shown modification in total protein concentration and variation in SDS-PAGE pattern. PS-6 was identified as Bacillus licheniformis based on partially sequenced of 16S rRNA using NCBI Blast. Further investigation will help in using this potential bacterium as a well-grounded source for urgency.

Keywords: arsenite, arsenate, Bacillus licheniformis, utilization

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Authors: Mahmoud M. Zakaria, Omnia F. Elmoursi, Mahmoud M. Gabr, Camelia A. AbdelMalak, Mohamed A. Ghoneim

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Many protocols were publicized for differentiation of human mesenchymal stem cells (MSCS) into insulin-producing cells (IPCs) in order to excrete insulin hormone ingoing to treat diabetes disease. Our aim is to evaluate relative gene expression for each independent protocol. Human bone marrow cells were derived from three volunteers that suffer diabetes disease. After expansion of mesenchymal stem cells, differentiation of these cells was done by three different protocols (the one-step protocol was used conophylline protein, the two steps protocol was depending on trichostatin-A, and the three-step protocol was started by beta-mercaptoethanol). Evaluation of gene expression was carried out by real-time PCR: Pancreatic endocrine genes, transcription factors, glucose transporter, precursor markers, pancreatic enzymes, proteolytic cleavage, extracellular matrix and cell surface protein. Quantitation of insulin secretion was detected by immunofluorescence technique in 24-well plate. Most of the genes studied were up-regulated in the in vitro differentiated cells, and also insulin production was observed in the three independent protocols. There were some slight increases in expression of endocrine mRNA of two-step protocol and its insulin production. So, the two-step protocol was showed a more efficient in expressing of pancreatic endocrine genes and its insulin production than the other two protocols.

Keywords: mesenchymal stem cells, insulin producing cells, conophylline protein, trichostatin-A, beta-mercaptoethanol, gene expression, immunofluorescence technique

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Authors: Mustafa Salman, Nurcan Cetinkaya, Zehra Selcuk, Bugra Genc

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The objectives of the present study were to estimate the microbial-N flow to the small intestine and to predict the digestible organic matter intake (DOMI) in grazing Karayaka sheep based on urinary excretion of purine derivatives (xanthine, hypoxanthine, uric acid, and allantoin) by the use of spot urine sampling under field conditions. In the trial, 10 Karayaka sheep from 2 to 3 years of age were used. The animals were grazed in a pasture for ten months and fed with concentrate and vetch plus oat hay for the other two months (January and February) indoors. Highly significant linear and cubic relationships (P<0.001) were found among months for purine derivatives index, purine derivatives excretion, purine derivatives absorption, microbial-N and DOMI. Through urine sampling and the determination of levels of excreted urinary PD and Purine Derivatives / Creatinine ratio (PDC index), microbial-N values were estimated and they indicated that the protein nutrition of the sheep was insufficient. In conclusion, the prediction of protein nutrition of sheep under the field conditions may be possible with the use of spot urine sampling, urinary excreted PD and PDC index. The mean purine derivative levels in spot urine samples from sheep were highest in June, July and October. Protein nutrition of pastured sheep may be affected by weather changes, including rainfall. Spot urine sampling may useful in modeling the feed consumption of pasturing sheep. However, further studies are required under different field conditions with different breeds of sheep to develop spot urine sampling as a model.

Keywords: Karayaka sheep, spot sampling, urinary purine derivatives, PDC index, microbial-N, feed intake

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Authors: Irena Kostovska, Katerina Tosheska Trajkovska, Svetlana Cekovska, Julijana Brezovska Kavrakova, Hristina Ampova, Sonja Topuzovska, Ognen Kostovski, Goce Spasovski, Danica Labudovic

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Background: Proteinuria is an important feature of secondary nephropathies. The quantitative and qualitative analysis of proteinuria plays an important role in determining the types of proteinuria (glomerular, tubular and mixed), in the diagnosis and prognosis of secondary nephropathies. The damage of the glomerular basement membrane is responsible for a proteinuria characterized by the presence of large amounts of protein with high molecular weights such as albumin (69 kilo Daltons-kD), transferrin (78 kD) and immunoglobulin G (150 kD). An insufficiency of proximal tubular function is the cause of a proteinuria characterized by the presence of proteins with low molecular weight (LMW), such as retinol binding protein (21 kD) and α1-microglobulin (31 kD). In some renal diseases, a mixed glomerular and tubular proteinuria is frequently seen. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is the most widely used method of analyzing urine proteins for clinical purposes. The main aim of the study is to determine the type of proteinuria in the most common secondary nephropathies such as diabetic, hypertensive nephropathy and preeclampsia. Material and methods: In this study were included 90 subjects: subjects with diabetic nephropathy (n=30), subjects with hypertensive nephropahty (n=30) and pregnant women with preeclampsia (n=30). We divided all subjects according to UM/CR into three subgroups: macroalbuminuric (UM/CR >300 mg/g), microalbuminuric (UM/CR 30-300 mg/g) and normolabuminuric (UM/CR<30 mg/g). In all subjects, we measured microalbumin and creatinine in urine with standard biochemical methods. Separation of urinary proteins was performed by SDS-PAGE, in several stages: linear gel preparation (4-22%), treatment of urinary samples before their application on the gel, electrophoresis, gel fixation, coloring with Coomassie blue, and identification of the separated protein fractions based on standards with exactly known molecular weight. Results: According to urinary microalbumin/creatinin ratio in group of subject with diabetic nephropathy, nine patients were macroalbuminuric, while 21 subject were microalbuminuric. In group of subjects with hypertensive nephropathy, we found macroalbuminuria (n=4), microalbuminuria (n=20) and normoalbuminuria (n=6). All pregnant women with preeclampsia were macroalbuminuric. Electrophoretic separation of urinary proteins showed that in macroalbuminric patients with diabetic nephropathy 56% have mixed proteinuria, 22% have glomerular proteinuria and 22% have tubular proteinuria. In subgroup of subjects with diabetic nephropathy and microalbuminuria, 52% have glomerular proteinuria, 8% have tubular proteinuria, and 40% of subjects have normal electrophoretic findings. All patients with maroalbuminuria and hypertensive nephropathy have mixed proteinuria. In subgroup of patients with microalbuminuria and hypertensive nephropathy, we found: 32% with mixed proteinuria, 27% with normal findings, 23% with tubular, and 18% with glomerular proteinuria. In all normoalbuminruic patiens with hypertensive nephropathy, we detected normal electrophoretic findings. In group of subjects pregnant women with preeclampsia, we found: 81% with mixed proteinuria, 13% with glomerular, and 8% with tubular proteinuria. Conclusion: By SDS PAGE method, we detected that in patients with secondary nephropathies the most common type of proteinuria is mixed proteinuria, indicating both loss of glomerular permeability and tubular function. We can conclude that SDS PAGE is high sensitive method for detection of renal impairment in patients with secondary nephropathies.

Keywords: diabetic nephropathy, preeclampsia, hypertensive nephropathy, SDS PAGE

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Authors: Moustafa Elhamouly, Masayuki Shimada

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The production of competent oocytes is essential for reproductivity in mammals. Maintenance of mitochondrial efficiency is required to supply the ATP necessary for granulosa cell proliferation during the follicular development process. Treatment with Pyrroloquinoline quinone (PQQ) has been reported to increase the number of ovulated oocytes and pups per delivery in mice by maintaining healthy mitochondrial function. This study aimed to elucidate how PQQ maintains mitochondrial function during ovarian follicle growth. To do this, both in vitro and in vivo experiments were performed with granulosa cells from superovulated immature (3-week-old) mice that were pretreated with or without PQQ. The effects of PQQ on beta-oxidation, mitochondrial function, mitophagy, and mitochondrial biogenesis were examined. PQQ increased beta-oxidation-related genes and CPT1 protein content in granulosa cells and this was associated with a decreased phosphorylation of P38 signaling protein. Using the fatty acid oxidation assay on the flux analyzer, PQQ increased the reliance of beta-oxidation on the endogenous fatty acids and was associated with a mild UCP-dependant mitochondrial uncoupling, ATP production, mitophagy, and mitochondrial biogenesis. PQQ also increased the expression of endogenous antioxidant enzymes. Thus, PQQ induced beta-oxidation in growing granulosa cells relying on endogenous fatty acids. And reduced the Reactive oxygen species (ROS) production by inducing a mild mitochondrial uncoupling with keeping high mitochondrial function. Damaged mitochondria were recycled by the induced mitophagy and replaced by the increased mitochondrial biogenesis. Collectively, PQQ may enhance reproductivity by maintaining the efficiency of mitochondria to produce enough ATP required for normal folliculogenesis.

Keywords: granulosa cells, mitochondrial uncoupling, mitophagy, pyrroloquinoline quinone (PQQ), reactive oxygen species (ROS).

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Authors: Michael P. Mannino, Gerald W. Hart

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The majority of glucose metabolism proceeds through glycolytic pathways such as glycolysis or pentose phosphate pathway, however, about 5% is shunted through the hexosamine biosynthetic pathway, producing uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). This precursor can then be incorporated into complex oligosaccharides decorating the cell surface or remain as an intracellular post-translational-modification (PTM) of serine/threonine residues (O-GlcNAcylation, OGN), which has been identified on over 4,000 cytosolic or nuclear proteins. Intracellular OGN has major implications on cellularprocesses, typically by modulating protein localization, protein-protein interactions, protein degradation, and gene expression. Additionally, OGN is known to have an extensive cross-talk with phosphorylation, be in a competitive or cooperative manner. Unlike other PTMs there are only two cycling enzymes that are capable of adding or removing the GlcNAc moiety, O-linked N-aceytl glucosamine Transferase (OGT) and O-linked N-acetyl glucoamidase (OGA), respectively. The activity of OGT has been shown to be sensitive to cellular UDP-GlcNAc levels, even changing substrate affinity. Owing to this and that the concentration of UDP-GlcNAc is related to the metabolisms of glucose, amino acid, fatty acid, and nucleotides, O-GlcNAc is often referred to as a nutrient sensing rheostat. Indeed OGN is known to regulate several signaling pathways as a result of nutrient levels, such as insulin signaling. Dysregulation of OGN is associated with several disease states such as cancer, diabetes, and neurodegeneration. Improvements in glycomics over the past 10-15 years has significantly increased the OGT substrate pool, suggesting O-GlcNAc’s involvement in a wide variety of signaling pathways. However, O-GlcNAc’s role at the receptor level has only been identified in a case-by-case basis of known pathways. Examining the OGN of the plasma membrane (PM) may better focus our understanding of O-GlcNAc-effected signaling pathways. In this current study, PM fractions were isolated from several cell types via ultracentrifugation, followed by purification and MS/MS analysis in several cell lines. This process was repeated with or without OGT/OGA inhibitors or with increased/decreased glucose levels in media to ascertain the importance of OGN. Various pathways are followed up on in more detailed studies employing methods to localize OGN at the PM specifically.

Keywords: GlcNAc, nutrient sensitive, post-translational-modification, receptor

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Authors: P. N. Okeke

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Fermentation and germination of legumes have been an ancient practice. In this study, the influence of fermentation and germination on the chemical properties of Lima bean (Phaseolus lunatus) flour were evaluated. The flours were analyzed for their proximate and mineral composition, using the standard assay methods. The result showed that fermentation and germination increased the moisture, protein and ash content of the flours while fiber, fat and carbohydrate were decreased. The protein level of fermented and germinated lima bean increased from 21.06–26.60%. The minerals: iron, copper, zinc, and phosphorous increased due to germination and fermentation. The phytate and tannin levels were drastically reduced in both the fermented and germinated flours. The result of this study revealed that fermentation and germination makes the nutrient in lima beans more accessible as it reduces the anti-nutrients. It is therefore recommended that lima bean be process accordingly for richer and more bio-availability of the nutrients.

Keywords: nutrient, anti-nutrient, fermented, germinated, lima bean flour

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Authors: M. K. Hossain, J. Keidel, O. Hensel, M. Diakite

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Fat reduced dairy products are holding a potential market due to health reason. Due to less creamy, and pleasantness, reduced and/or low-fat dairy products are getting less consumer acceptance whereas the fat molecule provides smooth, creamy and a pleasant mouthfeel in dairy products especially yogurt & ice cream. This study was aimed to investigate whether the application of microparticulated whey proteins (MWPs) processed by extrusion cooking, the reduced fat yogurt can achieve similar or higher creaminess compared to whole milk (3.8% fat) and skimmed milk (0.5% fat) yogurt. Full cream and skimmed milk were used to prepare natural stirred yogurt, as well as the dry matter content, also adjusted up to 16% with skimmed milk powder. Whey protein concentrates (WPC80) were used to produce MWPs in particle size of d50 > 5 µm, d50 3<5 µm and d50 < 3 µm through the hot-extrusion process with a screw speed of 400, 600 and 1000 rpm respectively. Furthermore, the commercially available microparticulated whey protein called Simplesse® was also applied in order to compare with extruded MWPs. The rheological and sensory properties of yogurt were assessed, and data were analyzed statistically. The applications of extruded MWPs with 600 and 1000 rpm were achieved significantly (p < 0.05) higher creaminess and preference compared to the whole and skimmed milk yogurt whereas, 400 rpm got lower preference. On the other hand, Simplesse® obtained the lowest creaminess and preference compared to other yogurts, although the contribution of dry matter in yogurt was same as extruded MWPs. The creaminess and viscosities were strongly (r = 0.62) correlated, furthermore, the viscosity from sensory evaluation and the dynamic viscosity of yogurt was also significantly (r = 0.72) correlated which clarifies that the performance of sensory panelists as well as the quality of the products.

Keywords: microparticulation, hot-extrusion, reduced-fat yogurt, whey protein concentrate

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Authors: L. Gómez-Limia, I. Franco, T. Blanco, S. Martínez

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European eels (Anguilla anguilla) belong to Anguilliformes order and Anguillidae family. They are generally classified as warm-water fish. Eels have a great commercial value in Europe and Asian countries. Eels can reach high weights, although their commercial size is relatively low in some countries. The capture of larger eels would facilitate the recovery of the species, as well as having a greater number of either glass eels or elvers for aquaculture. In the last years, the demand and the price of eels have increased significantly. However, European eel is considered critically endangered by the International Union for the Conservation of Nature (IUCN) Red List. The biochemical composition of fishes is an important aspect of quality and affects the nutritional value and consumption quality of fish. In addition, knowing this composition can help predict an individual’s condition for their recovery. Fish is known to be important source of protein rich in essential amino acids. However, there is very little information about changes in amino acids composition of European eels with increase in size. The aim of this study was to evaluate the effect of two different weight categories on the amino acids content in muscle tissue of wild European eels. European eels were caught in River Ulla (Galicia, NW Spain), during winter. The eels were slaughtered in ice water immersion. Then, they were purchased and transferred to the laboratory. The eels were subdivided into two groups, according to the weight. The samples were kept frozen (-20 °C) until their analysis. Frozen eels were defrosted and the white muscle between the head and the anal hole. was extracted, in order to obtain amino acids composition. Thirty eels for each group were used. Liquid chromatography was used for separation and quantification of amino a cids. The results conclude that the eels are rich in glutamic acid, leucine, lysine, threonine, valine, isoleucine and phenylalanine. The analysis showed that there are significant differences (p < 0.05) among the eels with different sizes. Histidine, threonine, lysine, hydroxyproline, serine, glycine, arginine, alanine and proline were higher in small eels. European eels muscle presents between 45 and 46% of essential amino acids in the total amino acids. European eels have a well-balanced and high quality protein source in the respect of E/NE ratio. However, eels with higher weight showed a better ratio of essential and non-essential amino acid.

Keywords: European eels, amino acids, HPLC, body size

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Authors: Hamim Abbas, Jean Luc-Hornick, Isabelle Dufrasne

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Feed costs constitute over 60% of total expenses in organic layer poultry production, with feed protein supply being a significant concern. Alfalfa-based dehydrated silage pellets are mainly diets composed of leaves (ABSP), which are non-conventional protein sources that could enhance profits by reducing feed costs and ensuring consistent availability. This experiment studied the effects on the performances of Novogen Brown light layers of a commercial control diet replaced with 10% ABSP. After a 21-day trial, this diet (ABSP) has improved the laying rate, yolk color of eggs, feed conversion rate, ω−3 (PUFAs) and ω−6/ω−3 ratio (P<0.05) while the body weight and egg weight were degraded with the substitution of the ABSP in the diet(P>0.05). The laying rate showed a tendency to increase (P=0.06). These findings suggest that ABSP can replace at least 10% of the feed in organic layer diets without compromising production parameters negatively.

Keywords: alfalfa, silage, pellet, organic layers

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Authors: Anjelika Kremer, Irina Nachimov, Dan Justo

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Background: C-reactive protein (CRP) serum levels are commonly measured in hospitalized patients. Elevated admission CRP serum levels and in-hospital mortality has been seldom studied in the general population of elderly patients admitted to the acute Geriatrics department. Methods: A retrospective cross-sectional study was conducted at a tertiary medical center. Included were all elderly patients (age 65 years or more) admitted to a single acute Geriatrics department from the emergency room between April 2014 and January 2015. CRP serum levels were measured routinely in all patients upon the first 24 hours of admission. A logistic regression analysis was used to study if admission CRP serum levels were associated with in-hospital mortality independent of age, gender, functional status, and co-morbidities. Results: Overall, 498 elderly patients were included in the analysis: 306 (61.4%) female patients and 192 (38.6%) male patients. The mean age was 84.8±7.0 years (median: 85 years; IQR: 80-90 years). The mean admission CRP serum levels was 43.2±67.1 mg/l (median: 13.1 mg/l; IQR: 2.8-51.7 mg/l). Overall, 33 (6.6%) elderly patients died during the hospitalization. A logistic regression analysis showed that in-hospital mortality was independently associated with history of stroke (p < 0.0001), heart failure (p < 0.0001), and admission CRP serum levels (p < 0.0001) – and to a lesser extent with age (p = 0.042), collagen vascular disease (p=0.011), and recent venous thromboembolism (p=0.037). Receiver operating characteristic (ROC) curve showed that admission CRP serum levels predict in-hospital mortality fairly with an area under the curve (AUC) of 0.694 (p < 0.0001). Cut-off value with maximal sensitivity and specificity was 19.7 mg/L. Conclusions: Admission CRP serum levels may be used to predict in-hospital mortality in the general population of elderly patients admitted to the acute Geriatrics department.

Keywords: c-reactive protein, elderly, mortality, prediction

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Authors: Wajad Nazeer, Saghir Ahmad, Khalid Mahmood, Altaf Hussain, Abid Mahmood, Baoliang Zhou

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MNH-886(Bt.) is a upland cotton cultivar (Gossypium hirsutum L.) developed through hybridization of three parents [(FH-207×MNH-770)×Bollgard-1] at Cotton Research Station Multan, Pakistan. It is resistant to CLCuVD with 16.25 % disease incidence (60 DAS, March sowing) whereas moderately susceptible to CLCuVD when planted in June with disease incidence 34 % (60 DAS). This disease reaction was lowest among 25 cotton advanced lines/varieties tested at hot spots of CLCuVD. Its performance was tested during 2009 to 2012 in various indigenous, provincial, and national varietal trials in comparison with the commercial variety IR-3701 and AA-802 & CIM-496. In PCCT trial during 2009-10; 2011-12, MNH-886 surpassed all the existing Bt. strains along with commercial varieties across the Punjab province with seed cotton yield production 2658 kg ha-1 and 2848 kg ha-1 which was 81.31 and 13% higher than checks, respectively. In National Coordinated Bt. Trial, MNH-886(Bt.) produced 3347 kg ha-1 seed cotton at CCRI, Multan; the hot spot of CLCuVD, in comparison to IR-3701 which gave 2556 kg ha-1. It possesses higher lint percentage (41.01%), along with the most desirable fibre traits (staple length 28.210mm, micronaire value 4.95 µg inch-1 and fibre strength 99.5 tppsi, and uniformity ratio 82.0%). The quantification of toxicity level of crystal protein was found positive for Cry1Ab/Ac protein with toxicity level 2.76µg g-1 and Mon 531 event was confirmed. Having tremendous yield potential, good fibre traits, and great tolerance to CLCuVD we can recommended this variety for cultivation in CLCuVD hotspots of Pakistan.

Keywords: cotton, cultivar, cotton leaf curl virus, CLCuVD hit districts

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Authors: Toyosi Yewande Tunde-Akintunde, Grace Oluwatoyin Ogunlakin, Bosede Folake Olanipekun

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Peppers are excellent sources of nutrients but its high moisture content makes it susceptible to spoilage. Drying, a common processing method, results in a reduction of these nutrients in the final product. Pre-treatment of pepper before drying can be used to reduce the level of degradation of nutrients. Thus this study investigated the effect of pre-treatment (hot water blanching and soaking in brine-sodium chloride) and drying methods (oven, microwave and sun) on selected quality parameters (proximate composition, capsaicin, reducing sugar and phenolic content, pH, total solid (TS), Titratable acidity (TA), water absorption capacity (WAC) and colour) of pepper. The protein and moisture content value ranged from 9.09 to 10.23% and 5.63 to 8.48% respectively. Sun dried samples had the highest value while oven dried samples had the lowest. Brine treated samples had higher protein but lower moisture content than blanched samples. Capsaicin, reducing sugar and phenolic content values ranged from 0.68 to 0.87 mg/dm3; 3.18 to 3.79 µg/ml; and 40.67 to 84.01 mg GAE/100 g d.m respectively. The sun dried samples had higher values while the lowest values were from microwave dried samples. The brine treated samples had higher values in capsaicin while the blanched samples had higher reducing sugar and phenolic contents. The values of L, a* and b* for the dried pepper varied from 58.76 to 63.13; 7.09 to 7.34; and 11.79 to 12.36 respectively. Oven dried samples had the lowest values for a*, while its L values were the highest. The L and a* values for brine treated samples were higher than blanched samples. The pre-treatment and drying method considered resulted in different values of the quality parameters considered which indicates that drying and pre-treatment has an effect on the quality of the final dried pepper samples.

Keywords: Bell pepper, microwave drying, oven drying, quality, sun drying

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Authors: Yeon Ho Je

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A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an_65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

Keywords: baculovirus, insecticide, neurotoxin, neurobactrus

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Authors: Ling-Ling E., Hong-Chen Liu, Dong-Sheng Wang, Fang Su, Xia Wu, Zhan-Ping Shi, Yan Lv, Jia-Zhu Wang

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Objective: The objective of the present study is to evaluate the capacity of a tissue-engineered bone complex of recombinant human bone morphogenetic protein 2 (rhBMP-2) mediated dental pulp stem cells (DPSCs) and nano-hydroxyapatite/collagen/poly(L-lactide)(nHAC/PLA) to reconstruct critical-size alveolar bone defects in New Zealand rabbit. Methods: Autologous DPSCs were isolated from rabbit dental pulp tissue and expanded ex vivo to enrich DPSCs numbers, and then their attachment and differentiation capability were evaluated when cultured on the culture plate or nHAC/PLA. The alveolar bone defects were treated with nHAC/PLA, nHAC/PLA+rhBMP-2, nHAC/PLA+DPSCs, nHAC/PLA+DPSCs+rhBMP-2, and autogenous bone (AB) obtained from iliac bone or were left untreated as a control. X-ray and a polychrome sequential fluorescent labeling were performed post-operatively and the animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis. Results: Our results showed that DPSCs expressed STRO-1 and vementin, and favoured osteogenesis and adipogenesis in conditioned media. DPSCs attached and spread well, and retained their osteogenic phenotypes on nHAC/PLA. The rhBMP-2 could significantly increase protein content, alkaline phosphatase (ALP) activity/protein, osteocalcin (OCN) content, and mineral formation of DPSCs cultured on nHAC/PLA. The X-ray graph, the fluorescent, histological observation and histomorphometric analysis showed that the nHAC/PLA+DPSCs+rhBMP-2 tissue-engineered bone complex had an earlier mineralization and more bone formation inside the scaffold than nHAC/PLA, nHAC/PLA+rhBMP-2 and nHAC/PLA+DPSCs, or even autologous bone. Implanted DPSCs contribution to new bone were detected through transfected eGFP genes. Conclutions: Our findings indicated that stem cells existed in adult rabbit dental pulp tissue. The rhBMP-2 promoted osteogenic capability of DPSCs as a potential cell source for periodontal bone regeneration. The nHAC/PLA could serve as a good scaffold for autologous DPSCs seeding, proliferation and differentiation. The tissue-engineered bone complex with nHAC/PLA, rhBMP-2, and autologous DPSCs might be a better alternative to autologous bone for the clinical reconstruction of periodontal bone defects.

Keywords: nano-hydroxyapatite/collagen/poly (L-lactide), dental pulp stem cell, recombinant human bone morphogenetic protein, bone tissue engineering, alveolar bone

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Authors: Natalia Moreno-Castellanos, Amaia Rodriguez, Gema Frühbeck

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Introduction: In adipocytes, the cytoskeleton undergoes important expression and distribution in adipocytes rearrangements during adipogenesis and in obesity. Indeed, a role for these proteins in the regulation of adipocyte differentiation and response to insulin has been demonstrated. Recently, septins have been considered as new components of the cytoskeletal network that interact with other cytoskeletal elements (actin and tubulin) profoundly modifying their dynamics. However, these proteins have not been characterized as yet in adipose tissue. In this work, were examined the cellular, molecular and functional features of a member of this family, septin 11 (SEPT11), in adipocytes and evaluated the impact of obesity on the expression of this protein in human adipose tissue. Methods: Adipose gene and protein expression levels of SEPT11 were analysed in human samples. SEPT11 distribution was evaluated by immunocytochemistry, electronic microscopy, and subcellular fractionation techniques. GST-pull down, immunoprecipitation and a Yeast-Two Hybrid (Y2H) screening were used to identify the SEPT11 interactome. Gene silencing was employed to assess the role of SEPT11 in the regulation of insulin signaling and lipid metabolism in adipocytes. Results: SEPT11 is expressed in human adipocytes, and its levels increased in both omental and subcutaneous adipose tissue in obesity, with SEPT11 mRNA content positively correlating with parameters of insulin resistance in subcutaneous fat. In non-stimulated adipocytes, SEPT11 immunoreactivity showed a ring-like distribution at the cell surface and associated to caveolae. Biochemical analyses showed that SEPT11 interacted with the main component of caveolae, caveolin-1 (CAV1) as well as with the fatty acid-binding protein, FABP5. Notably, the three proteins redistributed and co-localized at the surface of lipid droplets upon exposure of adipocytes to oleate. In this line, SEPT11 silencing in 3T3-L1 adipocytes impaired insulin signaling and decreased insulin-induced lipogenesis. Conclusions: Those findings demonstrate that SEPT11 is a novel component of the adipocyte cytoskeleton that plays an important role in the regulation of lipid traffic, metabolism and can thus represent a potential biomarker of insulin resistance in obesity in adipocytes through its interaction with both CAV1 and FABP5.

Keywords: caveolae, lipid metabolism, obesity, septins

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Authors: Rishi Saxena

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Cyanobacteria is a photosynthetic prokaryote, and these obtain their energy through photosynthesis. In this paper, we studied the effect of iron on metabolic changes in the diazotrophic cyanobacterium Anabaena PCC 7120. Nowadays, metal contamination due to natural and anthropogenic sources is a global environment concern. Iron induced changes in growth, N2-fixation, CO2 fixation and photosynthetic activity were studied in a diazotrophic cyanobacterium Anabaena PCC 7120. Iron at 50 uM concentration supported the maximum growth, heterocyst frequency, CO2 fixation, photosystem I (PS I), photosystem II (PS II) and nitrogenase activities in the organism. Higher concentration of iron inhibited these processes. Chl a and PS II activities were more sensitive to iron than the protein and PS I activity. Here, it is also mentioned that heavy metal induced altered macromolecules metabolism and changes in the central dogma of life (DNA→ mRNA → Protein). And also recent advances have been made in understanding heavy metal-cyanobacteria interaction and their application for metal detoxification.

Keywords: cyanobacterium anabaena 7120, nitrogen fixation, photosystem I (PS I), photosystem II (PS II)

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Authors: Mahsa Jalili, Essa Ehsan Khan, Signe Dille Lovmo, Augustine Akruwe, Egil Lien, Rolf Erik Olsen, Trygve Sigholt, Atle Magnus Bones

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Data from the Norwegian Directorate of Fisheries reported that >1.2 million tons of Atlantic salmon were produced in Norway aquaculture industry in 2016. Peroxisome proliferator-activated receptors (PPARs) are one of the key transcription factor families that respond to nutritional ligands. Recent studies have shown the connection between PPARs with lipid and carbohydrate metabolism in aquaculture. To our knowledge, there is no published data about the effects of krill meal, soybean meal, Bactocell ® and butyrate feedings compared to control group on PPARs gene and protein expressions in Atlantic salmon. Fish, 1year +postsmolt, average weight 250 gram were cultured for 12 weeks after acclimatization by control commercial feeding in 2 weeks after hatchery. Water oxygen rate, salinity, and temperature were monitored every second day. At the end of the trial, fish were taken from tanks randomly, and four replicates per group were collected and stored in -80 freezers until analysis. Total RNA extracted from posterior part of dorsal fin muscle tissues and Nanodrop and Bioanalyzer was used to check the quality of RNA. Gene expression of PPAR α, β and γ were determined by RT-PCR. The expression of genes of interest was measured relative to control group after normalization to three reference genes. Total protein concentration was calculated by Bradford method, and protein expression was determined with primary PPARγ antibody by western blot. All data were analyzed by ANOVA followed by Benjamini-Hochberg and Bonferroni tests. Probability values <0.05 considered significant. Bactocell® and butyrate groups showed significantly lower PPARα expression. PPARβ and γ were not significantly different among groups. PPARγ mRNA expression was approximately consistent with protein expression pattern, except than butyrate group showed lower mRNA level. The order of PPARγ expression was Bactocell® > soy meal > butyrate > krill meal > control respectively. PPARβ gene expression decreased more in soy meal > butyrate > krill meal > Bactocell® > control groups respectively. In conclusion, the increased expression of PPARγ and α is proposed to represent a reduction tendency of lipid storage in fish fed by Bactocell®, butyrate, soy and krill meal.

Keywords: aquaculture, blotting western, gene expression, krill protein extract, prebiotics, probiotics, Salmo salar

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Authors: Abdolamir Allameh, Shahnaz Esmaeili, Mina Allameh, Safoura Khajeniazi

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Stem cells with therapeutic applications can be isolated from human placenta/umblical cord blood (UCB) as well as the cord tissue (UC). Stem cells in culture are vulnerable to oxidative stress, particularly when subjected to differentiation process. The aim of this study was to examine the chnages in the rate of oxidation that occurs to cellular macromolecules during hepatic differentiation of mononuclear cells (MSCs). In addition, the impact of the hepatic differentiation process of MSC on cellular and biological activity of the cells will be undertaken. For this purpose, first mononuclear cells (MNCs) were isolated from human UCB which was obtained from a healthy full-term infant. The cells were cultured at a density of 3×10⁵ cells/cm² in DMEM- low-glucose culture media supplemented with 20% FBS, 2 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin. Cell cultures were then incubated at 37°C in a humidified 5% CO₂ incubator. After removing non-adherent cells by replacing culture medium, fibroblast-like adherent cells were resuspended in 0.25% trypsin-EDTA and plated in 25 cm² flasks (1×10⁴/ml). Characterization of the MSCs was routinely done by observing their morphology and growth curve. MSCs were subjected to a 2-step hepatocyte differentiation protocol in presence of hepatocyte growth factor (HGF), dexamethazone (DEX) and oncostatin M (OSM). The hepatocyte-like cells derived from MSCs were checked every week for 3 weeks for changes in lipid peroxidation, protein carbonyl formation and DNA oxidation i.e., 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assay. During the 3-week differentiation process of MSCs to hepatocyte-like cells we found that expression liver-specific markers such as albumin, was associated with increased levels of lipid peroxidation and protein carbonyl formation. Whereas, undifferentiated MSCs has relatively low levels of lipid peroxidation products. There was a significant increase ( p < 0.05) in lipid peroxidation products in hepatocytes on days 7, 14, and 21 of differentiation. Likewise, the level of protein carbonyls in the cells was elevated during the differentiation. The level of protein carbonyls measured in hepatocyte-like cells obtained 3 weeks after differentiation induction was estimated to be ~6 fold higher compared to cells recovered on day 7 of differentiation. On the contrary, there was a small but significant decrease in DNA damage marker (8-OH-dG) in hepatocytes recovered 3 weeks after differentiation onset. The level of 8-OHdG which was in consistent with formation of reactive oxygen species (ROS). In conclusion, this data suggest that despite the elevation in oxidation of lipid and protein molecules during hepatocyte development, the cells were normal in terms of DNA integrity, morphology, and biologically activity.

Keywords: adult stem cells, DNA integrity, free radicals, hepatic differentiation

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Authors: Filip Franciszek Karuga, Szymon Turkiewicz, Marta Ditmer, Marcin Sochal, Piotr Białasiewicz, Agata Gabryelska

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Circadian rhythm, an internal coordinator of physiological processes is composed of a set of semi-autonomous clocks. Clocks are regulated through the expression of circadian clock genes which form feedback loops, creating an oscillator. The primary loop consists of activators: CLOCK, BMAL1 and repressors: CRY, PER. CLOCK can be substituted by the Neuronal PAS Domain Protein 2 (NPAS2). Orphan nuclear receptor (REV-ERB-α) is a component of the secondary major loop, modulating the expression of BMAL1. Circadian clocks might be disrupted by the obstructive sleep apnea (OSA), which has also been associated with type II diabetes mellitus (DM2). Interestingly, studies suggest that dysregulation of NPAS2 and REV-ERB-α might contribute to the pathophysiology of DM2 as well. The goal of our study was to examine the role of NPAS2 and REV-ERB-α in DM2 in OSA patients. After examination of the clinical data, all participants underwent polysomnography (PSG) to assess their apnea-hypopnea index (AHI). Based on the acquired data participants were assigned to one of 3 groups: OSA (AHI>30, no DM2; n=17 for NPAS2 and 34 for REV-ERB-α), DM2 (AHI>30 + DM2; n=7 for NPAS2 and 15 for REV-ERB-α) and control group (AHI<5, no DM2; n=16 for NPAS2 and 31 for REV-ERB-α). ELISA immunoassay was performed to assess the serum protein level of REV-ERB-α and NPAS2. The only statistically significant difference between groups was observed in NPAS2 protein level (p=0.037). Post-hoc analysis showed significant differences between the OSA and the control group (p=0.017). AHI and NPAS2 level was significantly correlated (r=-0.478, p=0.002) in all groups. A significant correlation was observed between the REV-ERB-α level and sleep efficiency (r=0.617, p=0.005) as well as sleep maintenance efficiency (r=0.645, p=0.003) in the OSA group. We conclude, that NPAS2 is associated with OSA severity and might contribute to metabolic sequelae of this disease. REV-ERB-α on the other hand can influence sleep continuity and efficiency.

Keywords: OSA, diabetes mellitus, endocrinology, chronobiology

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Authors: Weikang Gong, Chunhua Li

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Dynamics plays an essential role in function exertion of proteins. Elastic network model (ENM), a harmonic potential-based and cost-effective computational method, is a valuable and efficient tool for characterizing the intrinsic dynamical properties encoded in biomacromolecule structures and has been widely used to detect the large-amplitude collective motions of proteins. Gaussian network model (GNM) and anisotropic network model (ANM) are the two often-used ENM models. In recent years, many ENM variants have been proposed. Here, we propose a small but effective modification (denoted as modified mENM) to the multiscale ENM (mENM) where fitting weights of Kirchhoff/Hessian matrixes with the least square method (LSM) is modified since it neglects the details of pairwise interactions. Then we perform its comparisons with the original mENM, traditional ENM, and parameter-free ENM (pfENM) on reproducing dynamical properties for the six representative proteins whose molecular dynamics (MD) trajectories are available in http://mmb.pcb.ub.es/MoDEL/. In the results, for B-factor prediction, mENM achieves the best performance among the four ENM models. Additionally, it is noted that with the weights of the multiscale Kirchhoff/Hessian matrixes modified, interestingly, the modified mGNM/mANM still has a much better performance than the corresponding traditional ENM and pfENM models. As to dynamical cross-correlation map (DCCM) calculation, taking the data obtained from MD trajectories as the standard, mENM performs the worst while the results produced by the modified mENM and pfENM models are close to those from MD trajectories with the latter a little better than the former. Generally, ANMs perform better than the corresponding GNMs except for the mENM. Thus, pfANM and the modified mANM, especially the former, have an excellent performance in dynamical cross-correlation calculation. Compared with GNMs (except for mGNM), the corresponding ANMs can capture quite a number of positive correlations for the residue pairs nearly largest distances apart, which is maybe due to the anisotropy consideration in ANMs. Furtherly, encouragingly the modified mANM displays the best performance in capturing the functional motional modes, followed by pfANM and traditional ANM models, while mANM fails in all the cases. This suggests that the consideration of long-range interactions is critical for ANM models to produce protein functional motions. Based on the analyses, the modified mENM is a promising method in capturing multiple dynamical characteristics encoded in protein structures. This work is helpful for strengthening the understanding of the elastic network model and provides a valuable guide for researchers to utilize the model to explore protein dynamics.

Keywords: elastic network model, ENM, multiscale ENM, molecular dynamics, parameter-free ENM, protein structure

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Authors: Marina Lazar Chandy, N. Kannan, Rajendra Patil, Vinod Mathew, Ajmal Mohamed, P. K. Sreeja, Renju Jose

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Introduction- Arecoline, a major constituent of arecanut has shown to have some effect on liver. The use of arecanut is found to be the most common etiological factor for the development of Oral Submucous fibrosis (O.S.M.F). The effect of arecanut usage on liver in patients with O.S.M.F needs to be assessed. Lipids play a role in structural maintenance of cell. Alterations of lipid profile were noted in cancer patients. O.S.M.F being a precancerous lesion can have some effect on the level of lipids in the body. Objectives: This study was done to assess the alterations in liver enzymes (Serum Glutamic Pyruvic Transaminase(S.G.P.T ,Serum Glutamic Oxaloacetic Transaminase(S.G.O.T)) and lipid metabolism (High Density Lipoprotien(H.D.L) and Apo Lipoprotien A1 (Apo A1)) in patients with O.S.M.F. Methods-130 patients were taken for the study,100 patients with O.S.M.F and 30 as control group without O.S.M.F. Fasting blood sugar levels were taken, centrifuged and analyzed for S.G.P.T,S.G.O.T, H.D.L and Apo A1 using semi automated spectrophotometer. Results: After statistical analysis, it was concluded that there is an elevation of levels of S.G.P.T, S.G.O.T, and decreased levels of H.D.L, Apo A1 for O.S.M.F group when compared with control group. With increased grade of O.S.M.F. and duration of habit, S.G.P.T. & S.G.O.T. increased whereas, H.D.L. & Apo A1 decreased. All the values were statistically significant at p<0.01.

Keywords: apolipoprotien A1, high density lipoprotien, oral submucous fibrosis, serum glutamic oxaloacetic transaminase

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Authors: Dake Xiong, Ben Hankamer, Ian Ross

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The most urgent challenge of our time is to replace the depleting resources of fossil fuels by sustainable environmentally friendly alternatives. Hydrogen is a promising CO2-neutral fuel for a more sustainable future especially when produced photo-biologically. Hydrogen can be photosynthetically produced in unicellular green alga like Chlamydomonas reinhardtii, catalysed by the inducible highly active and bidirectional [FeFe]-hydrogenase enzymes (HydA). However, evolutionary and physiological constraints severely restrict the hydrogen yield of algae for industrial scale-up, mainly due to its competition among other metabolic pathways on photosynthetic electrons. Among them, a major challenge to be resolved is the inferior competitiveness of hydrogen production (catalysed by HydA) with NADPH production (catalysed by ferredoxin-NADP+-reductase (FNR)), which is essential for cell growth and takes up ~95% of photosynthetic electrons. In this work, the in vivo hydrogen production efficiency of mutants with ferredoxin-hydrogenase (Fd*-HydA1*) fusion protein construct, where the electron donor ferredoxin (Fd*) is fused to HydA1* and expressed in the model organism C. reinhardtii was investigated. Once Fd*-HydA1* fusion gene is expressed in algal cells, the fusion enzyme is able to draw the redistributed photosynthetic electrons and use them for efficient hydrogen production. From preliminary data, mutants with Fd*-HydA1* transgene showed a ~2-fold increase in the photosynthetic hydrogen production rate compared with its parental strain, which only possesses the native HydA in vivo. Therefore, a solid method of having more efficient hydrogen production in microalgae can be achieved through the expression of the synthetic enzymes.

Keywords: Chlamydomonas reinhardtii, ferredoxin, fusion protein, hydrogen production, hydrogenase

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Authors: Goksen Capar, Levent Dogankaya

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According to the circular economy model, resource productivity should be maximized and wastes should be reduced. Since earth’s natural resources are continuously depleted, resource recovery has gained great interest in recent years. As part of our research study on the recovery and reuse of silk wastes, this paper focuses on the utilization of silkworm pupae as fishmeal replacement, which would replace the original fishmeal raw material, namely the fish itself. This, in turn, would contribute to sustainable management of wild fish resources. Silk fibre is secreted by the silkworm Bombyx mori in order to construct a 'room' for itself during its transformation process from pupae to an adult moth. When the cocoons are boiled in hot water, silk fibre becomes loose and the silk yarn is produced by combining thin silk fibres. The remaining wastes are 1) sericin protein, which is dissolved in water, 2) remaining part of cocoon, including the dead body of B. mori pupae. In this study, an eight weeks trial was carried out to determine the growth performance of common carp juveniles fed with waste silkworm pupae meal (SWPM) as a replacement for fishmeal (FM). Four isonitrogenous diets (40% CP) were prepared replacing 0%, 33%, 50%, and 100% of the dietary FM with non-defatted silkworm pupae meal as a dietary protein source for experiments in C. carpio. Triplicate groups comprising of 20 fish (0.92±0.29 g) were fed twice/day with one of the four diets. Over a period of 8 weeks, results showed that the diet containing 50% of its protein from SWPM had significantly higher (p ≤ 0.05) growth rates in all groups. The increasing levels of SWPM were resulted in a decrease in growth performance and significantly lower growth (p ≤ 0.05) was observed with diets having 100% SWPM. The study demonstrates that it is practical to replace 50% of the FM protein with SWPM with a significantly better utilization of the diet but higher SWPM levels are not recommended for juvenile carp. Further experiments are under study to have more detailed results on the possible effects of this alternative diet on the growth performance of juvenile carp.

Keywords: Bombyx mori, Cyprinus carpio, fish meal, silk, waste pupae

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Authors: Dong-Gyu Leem, Ji-Sun Shin, Sang Yoon Choi, Kyung-Tae Lee

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In this study, we focused on the anti-proliferative effects of panaxydol, a C17 polyacetylenic compound derived from Panax ginseng roots, against various human cancer cells. We treated with panaxydol to various cancer cells and panaxydol treatment was found to significantly inhibit the proliferation of human lung cancer cells (A549) and human pancreatic cancer cells (AsPC-1 and MIA PaCa-2), of which AsPC-1 cells were most sensitive to its treatment. DNA flow cytometric analysis indicated that panaxydol blocked cell cycle progression at the G1 phase in A549 cells, which accompanied by a parallel reduction of protein expression of cyclin-dependent kinase (CDK) 2, CDK4, CDK6, cyclin D1 and cyclin E. CDK inhibitors (CDKIs), such as p21CIP1/WAF1 and p27KIP1, were gradually upregulated after panaxydol treatment at the protein levels. Furthermore, panaxydol induced the activation of p53 in A549 cells. In addition, panaxydol also induced apoptosis of AsPC-1 and MIA PaCa-2 cells, as shown by accumulation of subG1 and apoptotic cell populations. Panaxydol triggered the activation of caspase-3, -8, -9 and the cleavage of poly (ADP-ribose) polymerase (PARP). Reduction of mitochondrial transmembrane potential by panaxydol was determined by staining with dihexyloxacarbocyanine iodide. Furthermore, panaxydol suppressed the levels of anti-apoptotic proteins, XIAP and Bcl-2, and increased the levels of proapoptotic proteins, Bax and Bad. In addition, panaxydol inhibited the activation of Akt and extracellular signal-regulated kinase (ERK) and activated the p38 mitogen-activated protein kinase kinase (MAPK). Our results suggest that panaxydol is an anti-tumor compound that causes p53-mediated cell cycle arrest and apoptosis via mitochondrial apoptotic pathway in various cancer cells.

Keywords: apoptosis, cancer, G1 arrest, panaxydol

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Authors: Farahnaz Sadat Golestan Hashemi, Mohd Razi Ismail, Mohd Y. Rafii

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Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system can facilitate targeted genome editing in organisms. Dual or single guide RNA (gRNA) can program the Cas9 nuclease to cut target DNA in particular areas; thus, introducing concise mutations either via error-prone non-homologous end-joining repairing or via incorporating foreign DNAs by homologous recombination between donor DNA and target area. In spite of high demand of such promising technology, developing a well-organized procedure in order for reliable mining of potential target sites for gRNAs in large genomic data is still challenging. Hence, we aimed to perform high-throughput detection of target sites by specific PAMs for not only common Streptococcus pyogenes (SpCas9) but also for Neisseria meningitides (NmCas9) CRISPR-Cas systems. Previous research confirmed the successful application of such RNA-guided Cas9 orthologs for effective gene targeting and subsequently genome manipulation. However, Cas9 orthologs need their particular PAM sequence for DNA cleavage activity. Activity levels are based on the sequence of the protospacer and specific combinations of favorable PAM bases. Therefore, based on the specific length and sequence of PAM followed by a constant length of the target site for the two orthogonals of Cas9 protein, we created a reliable procedure to explore possible gRNA sequences. To mine CRISPR target sites, four different searching modes of sgRNA binding to target DNA strand were applied. These searching modes are as follows i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching, and iv) paired-gRNA searching. Finally, a complete list of all potential gRNAs along with their locations, strands, and PAMs sequence orientation can be provided for both SpCas9 as well as another potential Cas9 ortholog (NmCas9). The artificial design of potential gRNAs in a genome of interest can accelerate functional genomic studies. Consequently, the application of such novel genome editing tool (CRISPR/Cas technology) will enhance by presenting increased versatility and efficiency.

Keywords: CRISPR/Cas9 genome editing, gRNA mining, SpCas9, NmCas9

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Authors: Xiaolin Li, Terence Turney, John Forsythe, Bryce Feltis, Paul Wright, Vinh Truong, Will Gates

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Clay-hydrogel nanocomposites have attracted great attention recently, mainly because of their enhanced mechanical properties and ease of fabrication. Moreover, the unique platelet structure of clay nanoparticles enables the incorporation of bioactive molecules, such as proteins or drugs, through ion exchange, adsorption or intercalation. This study seeks to improve the mechanical and rheological properties of a novel hydrogel system, copolymerized from a tetrapodal polyethylene glycol (PEG) thiol and a linear, triblock PEG-PPG-PEG (PPG: polypropylene glycol) α,ω-bispropynoate polymer, with the simultaneous incorporation of various amounts of Na-saturated, montmorillonite clay (MMT) platelets (av. lateral dimension = 200 nm), to form a bioactive three-dimensional network. Although the parent hydrogel has controlled swelling ability and its PEG groups have good affinity for the clay platelets, it suffers from poor mechanical stability and is currently unsuitable for potential applications. Nanocomposite hydrogels containing 4wt% MMT showed a twelve-fold enhancement in compressive strength, reaching 0.75MPa, and also a three-fold acceleration in gelation time, when compared with the parent hydrogel. Interestingly, clay nanoplatelet incorporation into the hydrogel slowed down the rate of its dehydration in air. Preliminary results showed that protein binding by the MMT varied with the nature of the protein, as horseradish peroxidase (HRP) was more strongly bound than bovine serum albumin. The HRP was no longer active when bound, presumably as a result of extensive structural refolding. Further work is being undertaken to assess protein binding behaviour within the nanocomposite hydrogel for potential diabetic wound healing applications.

Keywords: hydrogel, nanocomposite, small molecule, wound healing

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Authors: Alireza Jalalvand, Maryam Saleh, Somayeh Behjat Khatouni, Zahra Bahri Najafi, Foroozan Fatahinia, Narges Ismailzadeh, Behrokh Farahmand

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Object: In the last two decades, the recent outbreak of Coronavirus (SARS-CoV-2) imposed a global pandemic in the world. Despite the increasing prevalence of the disease, there are no effective drugs to treat it. A suitable and rapid way to afford an effective drug and treat the global pandemic is a computational drug study. This study used molecular docking methods to examine the potential inhibition of over 50 antiviral drugs against three fundamental proteins of SARS-CoV-2. METHODS: Through a literature review, three important proteins (a key protease, RNA-dependent RNA polymerase (RdRp), and spike) were selected as drug targets. Three-dimensional (3D) structures of protease, spike, and RdRP proteins were obtained from the Protein Data Bank. Protein had minimal energy. Over 50 antiviral drugs were considered candidates for protein inhibition and their 3D structures were obtained from drug banks. The Autodock 4.2 software was used to define the molecular docking settings and run the algorithm. RESULTS: Five drugs, including indinavir, lopinavir, saquinavir, nelfinavir, and remdesivir, exhibited the highest inhibitory potency against all three proteins based on the binding energies and drug binding positions deduced from docking and hydrogen-bonding analysis. Conclusions: According to the results, among the drugs mentioned, saquinavir and lopinavir showed the highest inhibitory potency against all three proteins compared to other drugs. It may enter laboratory phase studies as a dual-drug treatment to inhibit SARS-CoV-2.

Keywords: covid-19, drug repositioning, molecular docking, lopinavir, saquinavir

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