Search results for: scaffold protein

Authors: Sulhee Lee, Geon Kim, Young-Seo Park

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Unlike the traditional milk sterilization methods (LTLT, HTST, or UHT), pulsed electric field (PEF) technology is a non-thermal pasteurization process. This technology minimizes energy required for heat treatment in food processing, changes in sensory properties, and physical losses. In this study, structural changes of bovine milk proteins, the amount of immunoproteins such as IgG, and their storability by PEF treatment were examined. When the changes of protein content in PEF-treated milk were examined using HPLC, the amounts of α-casein and β-lactoglobulin were reduced over 40% each, whereas those of κ-casein and β-casein did not change. The amount of α-casein in HTST milk was reduced to 50%. When PEF was applied to milk at the energy level of 250 kJ, the amounts of IgG, IgA, β-lactoglobulin (β-LG), lactoferrin, and α-lactalbumin (α-LA) decreased by 43, 41, 35, 63, and 45%, respectively. When milk was sterilized by LTLT process followed by PEF process at the level of 150 kJ, the concentrations of IgG, IgA, β-LG, lactoferrin, and α-LA were 56.6, 10.6, 554, 2.8 and 660.1 μg/mL, respectively. When the bovine milk was sterilized by LTLT process followed by PEF process at the energy level of 180 kJ, storability of immunoproteins of milk was the highest and the concentrations of IgG, IgA, and β-LG decreased by 79.5, 6.5, and 134.5 μg/mL, respectively, when compared with the initial concentrations of those proteins. When bovine milk was stored at 4℃ after sterilization through HTST sterilizer followed by PEF process at the energy level of 200 kJ, the amount of lactoferrin decreased 7.3% after 36 days of storage, whereas that of lactoferrin of raw milk decreased 16.4%. Our results showed that PEF treatment did not change the protein structure nor induce protein denaturation in milk significantly when compared with LTLT or HTST sterilization. Also, LTLT or HTST process in combination with PEF were more effective than LTLT only or HTST only process in the conservation of immunoproteins in bovine milk.

Keywords: pulsed electric field, bovine milk, immunoproteins, sterilization

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Authors: Elif Gencturk, Ekin Yurdakul, Ahmet Y. Celik, Senol Mutlu, Kutlu O. Ulgen

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Yeast cells are generally used as a model system of eukaryotes due to their complex genetic structure, rapid growth ability in optimum conditions, easy replication and well-defined genetic system properties. Thus, yeast cells increased the knowledge of the principal pathways in humans. During fermentation, carbohydrates (hexoses and pentoses) degrade into some toxic by-products such as 5-hydroxymethylfurfural (5-HMF or HMF) and furfural. HMF influences the ethanol yield, and ethanol productivity; it interferes with microbial growth and is considered as a potent inhibitor of bioethanol production. In this study, yeast single cell behavior under HMF application was monitored by using a continuous flow single phase microfluidic platform. Microfluidic device in operation is fabricated by hot embossing and thermo-compression techniques from cyclo-olefin polymer (COP). COP is biocompatible, transparent and rigid material and it is suitable for observing fluorescence of cells considering its low auto-fluorescence characteristic. The response of yeast cells was recorded through Red Fluorescent Protein (RFP) tagged Nop56 gene product, which is an essential evolutionary-conserved nucleolar protein, and also a member of the box C/D snoRNP complexes. With the application of HMF, yeast cell proliferation continued but HMF slowed down the cell growth, and after HMF treatment the cell proliferation stopped. By the addition of fresh nutrient medium, the yeast cells recovered after 6 hours of HMF exposure. Thus, HMF application suppresses normal functioning of cell cycle but it does not cause cells to die. The monitoring of Nop56 expression phases of the individual cells shed light on the protein and ribosome synthesis cycles along with their link to growth. Further computational study revealed that the mechanisms underlying the inhibitory or inductive effects of HMF on growth are enriched in functional categories of protein degradation, protein processing, DNA repair and multidrug resistance. The present microfluidic device can successfully be used for studying the effects of inhibitory agents on growth by single cell tracking, thus capturing cell to cell variations. By metabolic engineering techniques, engineered strains can be developed, and the metabolic network of the microorganism can thus be manipulated such that chemical overproduction of target metabolite is achieved along with the maximum growth/biomass yield.  

Keywords: COP, HMF, ribosome biogenesis, thermoplastic microbioreactor, yeast

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Authors: Hainan Chen, Jina Qing, Xiao Zhu, Ling Gao, Ampadu O. Jackson, Min Zhang, Kai Yin

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Objective: Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is highly associated with mitochondrial respiration. Recent studies have suggested that mitochondrial apolipoprotein A-1 binding protein (APOA1BP) was essential for the cellular metabolite NADHX repair to NADH, which is necessary for the mitochondrial function. The exact role of APOA1BP in the repolarization of M1 to M2, however, is uncertain. Material and method: THP-1-derived macrophages were incubated with LPS (10 ng/ml) or/and IL-4 (100 U/ml) for 24 hours. Biochemical parameters of oxidative phosphorylation and M1/M2 markers were analyzed after overexpression of APOA1BP in cells. Results: Compared with control and IL-4-exposed M2 cells, APOA1BP was downregulated in M1 macrophages. APOA1BP restored the decline in mitochondrial function to improve metabolic and phenotypic reprogramming of M1 to M2 macrophages. Blocking oxidative phosphorylation by oligomycin blunts the effects of APOA1BP on M1 to M2 repolarization. Mechanistically, LPS triggered the hydration of NADH and increased its hydrate NADHX which inhibit cellular NADH dehydrogenases, a key component of electron transport chain for oxidative phosphorylation. APOA1BP decreased the level of NADHX via converting R-NADHX to biologically useful S-NADHX. The mutant of APOA1BP aspartate188, the binding site of NADHX, fail to repair oxidative phosphorylation, thereby preventing repolarization. Conclusions: Restoring mitochondrial function by increasing mitochondrial APOA1BP might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control inflammatory diseases.

Keywords: inflammatory diseases, macrophage repolarization, mitochondrial respiration, apolipoprotein A-1 binding protein, NADHX, NADH

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Authors: Zeiler M., Stadler D., Schmaderer C.

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Over the years of dialysis treatment, most patients experience significant weight loss. The primary emphasis in earlier research was the underlying mechanism of protein energy wasting and the subsequent malnutrition inflammation syndrome. In the interest to provide an effective and rapid solution for the patients, the aim of this study is identifying individual influences of their assumed reduced dietary intake, such as nausea, appetite loss and taste changes, and to determine whether the patients adhere to their nutrition guidelines. A prospective, controlled study with 38 end-stage renal disease patients was performed using a questionnaire to reflect their diet within the last 12 months. Thereby, the daily intake for the most important macro-and micronutrients was calculated to be compared with the individual KDQOI-guideline value, as well as controls matched in age and gender. The majority of the study population did not report symptoms commonly associated with dialysis, such as nausea or inappetence, and denied any change in dietary behavior since receiving renal replacement therapy. The patients’ daily intake of energy (3080kcal ± 1266) and protein (89,9g [53,4-142,0]) did not differ significantly from the controls (energy intake: 3233kcal ± 1046, p=0,597; protein intake: 103,7g [90,1-125,5], p=0,120). The average difference to the individual calculated KDQOI-guideline was +176,0kcal ± 1156 (p=0,357) for energy intake and -1,75g ± 45,9 (p=0,491) for protein intake. However, there was an observed imbalance in the distribution of macronutrients, with a preference for fats over proteins. The patients’ daily intake of sodium (5,4g [ 2,95-10,1]) was higher than in the controls (4,1g [2,04-5,99], p= 0,058) whereas both values for potassium (3,7g ± 1,84) and phosphorous (1,79g ± 0,91) went significantly below the controls’ values (potassium intake: 4,89g ± 1,74, p=0,014; phosphorous intake: 2,04g ± 0,64, p=0,038). Thus, the values exceeded the calculated KDQOI-recommendation by + 3,3g [0,63-7,90] (p<0,001) for sodium, +1,49g ± 1,84 (p<0,001) for potassium and +0,89g ± 0,91 (p<0,001) for phosphorous. Contrary to the assumption, the patients did not under-eat. Nevertheless, their diets did not align with the recommended values. These findings highlight the need for intervention and education among patients and that regular dietary monitoring could prevent unhealthy nutrition habits. The elaboration of individual references instead of standardized guidelines could increase the compliance to the advised diet so that interdisciplinary comorbidities do not develop or worsen.

Keywords: compliance, dialysis, end-stage renal disease, KDQOI, malnutrition, nutrition guidelines, questionnaire, salt intake

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Authors: Ajith K. A. Gedara, Iva Chianella, Jose L. Endrino, Qi Zhang

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The olive seed is a by-product of the olive oil production industry. Biuret test and ferric chloride test revealed that water or alkali NaOH extractions of olive seed flour are rich in proteins and tannins. Both protein and tannins are well-known bio-based wood adhesives in the wood-based panel industry. In general, tannins-based adhesives show better mechanical and physical properties than protein wood adhesives. This paper explores different methods of extracting tannins from olive seed flour against the tannins yield and their applications as bio-based adhesives in wood-based panels. Once investigated, the physical and the mechanical properties of wood-based panels made using bio-adhesives based tannins extracted from olive seed flour revealed that the resulting products seemed to satisfy the Japanese Industrial Standards JIS A 5908:2015.

Keywords: bio-adhesives, olive seed flour, tannins, wood-based panels

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Authors: J. Adeppa, S. Samanta, O. K. Raina

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Fasciolosis is a widespread economically important parasitic infection throughout the world caused by Fasciola hepatica and F. gigantica. In order to identify novel immunogen conferring significant protection against fasciolosis, currently, research has been focused on the defined antigens viz. glutathione S-transferase, fatty acid binding protein, cathepsin-L, fluke hemoglobin, paramyosin, myosin and F. hepatica- Kunitz Type Molecule. Among various antigens, GST which plays a crucial role in detoxification processes, i.e. phase II defense mechanism of this parasite, has a unique position as a novel vaccine candidate and a drug target in the control of this disease. For producing the antigens in large quantities and their purification to complete homogeneity, the recombinant DNA technology has become an important tool to achieve this milestone. RT- PCR was carried out using F. gigantica total RNA as template, and an amplicon of 657 bp GST gene was obtained. TA cloning vector was used for cloning of this gene, and the presence of insert was confirmed by blue-white selection for recombinant colonies. Sequence analysis of the present isolate showed 99.1% sequence homology with the published sequence of the F. gigantica GST gene of cattle origin (accession no. AF112657), with six nucleotide changes at 72, 74, 423, 513, 549 and 627th bp found in the present isolate, causing an overall change of 4 amino acids. The 657 bp GST gene was cloned at BamH1 and HindIII restriction sites of the prokaryotic expression vector pPROEXHTb in frame with six histidine residues and expressed in E. coli DH5α. Recombinant protein was purified from the bacterial lysate under non-denaturing conditions by the process of sonication after lysozyme treatment and subjecting the soluble fraction of the bacterial lysate to Ni-NTA affinity chromatography. Western blotting with rabbit hyper-immune serum showed immuno-reactivity with 25 kDa recombinant GST. Recombinant protein detected F. gigantica experimental as well as field infection in buffaloes by dot-ELISA. However, cross-reactivity studies on Fasciola gigantica GST antigen are needed to evaluate the utility of this protein in the serodiagnosis of fasciolosis.

Keywords: fasciola gigantic, fasciola hepatica, GST, RT- PCR

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Authors: Sarah Elsayed Mohammed, Lamiaa Ahmed Ahmed, Mahmoud Mohammed Khattab

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Aim: The aim of the present study was to investigate whether combined nicorandil and bone marrow-derived mesenchymal stem cells (BMDMSC) treatment could offer an additional benefit in ameliorating isoproterenol (ISO)-induced heart failure in rats. Methods: ISO (85 and 170 mg/kg/day) was injected subcutaneously for 2 successive days, respectively. By day 3, electrocardiographic changes were recorded and serum was separated for determination of CK-MB level for confirmation of myocardial damage. Nicorandil (3 mg/kg/day) was then given orally with or without a single i.v. BMDMSC administration. Electrocardiography and echocardiography were recorded 2 weeks after beginning of treatment. Rats were then sacrificed and ventricles were isolated for estimation of vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) contents, caspase-3 activity as well as inducible nitric oxide synthase (iNOS) and connexin-43 protein expressions. Moreover, histological analysis of myocardial fibrosis was performed and cryosections were done for estimation of homing of BMDMSC. Results: ISO induced a significant increase in ventricles/body weight ratio, left ventricular end diastolic (LVEDD) and systolic dimensions (LVESD), ST segment and QRS duration. Moreover, myocardial fibrosis as well as VEGF, TNF-α and TGF-β contents were significantly increased. On the other hand, connexin-43 protein expression was significantly decreased, while caspase-3 and iNOS protein expressions were significantly increased. Combined therapy provided additional improvement compared to cell treatment alone towards reducing cardiac hypertrophy, fibrosis and inflammation. Furthermore, combined therapy induced significant increase in angiogenesis and BMDMSC homing and prevented ISO induced changes in iNOS, connexin-43 and caspase-3 protein expressions. Conclusion: Combined nicorandil/BMDMSC treatment was superior to BMDMSC alone towards preventing ISO-induced heart failure in rats.

Keywords: fibrosis, isoproterenol, mesenchymal stem cells, nicorandil

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Authors: Nurcan Cetinkaya, Nadide Hulya Ozdemir

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The aim of the study was to investigate the effect of Saccharomyces cerevisiae (SC) live yeast culture on microbial protein supply to the small intestine in Kivircik male yearlings when fed with different ratio of forage and concentrate diets. Four Kivircik male yearlings with permanent rumen canula were used in the experiment. . The treatments were allocated to a 4x4 Latin square design. Diet I consisted of 70% alfalfa hay and 30% concentrate, Diet II consisted of 30% alfalfa hay and 70% concentrate, Diet I and II were supplemented with a SC. Daily urine was collected and stored at -20°C until analysis. Calorimetric methods were used for the determination of urinary allantoin and creatinin levels. The estimated microbial N supply to small intestine for Diets I, I+SC, II and II+SC were 2.51, 2.64, 2.95 and 3.43 g N/d respectively. Supplementation of Diets I and II with SC significantly affected the allantoin levels in µmol/W0. 75 (p<0.05). Mean creatinine values in µmol/W0. 75 and allantoin:creatinin ratios were not significantly different among diets. In conclusion, supplementation with SC live yeast culture had a significant effect on urinary allantoin excretion and microbial protein supply to small intestine in Kivircik yearlings fed with high concentrate Diet II (P<0.05). Hence urinary allantoin excretion may be used as a tool for estimating microbial protein supply in Kivircık yearlings. However, further studies are necessary to understand the metabolism of Saccharomyces cerevisiae live yeast culture with different forage: concentrate ratio in Kıvırcık Yearlings.

Keywords: allantoin, creatinin, Kivircik yearling, microbial nitrogen, Saccharomyces cerevisia

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Authors: Sonaal Ramsookmohan

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Legume seeds are common foods in human diet and have been identied as a valuable source of human nutritonn Since they are useful sources of protein; legume proteins are used in many food applicatonsn Critcal functonal propertes are recognized to impact the quality of foodn Cowpea (Vigna unguiculata), has been well documented for its immense potental in contributng to food security forming part of daily staple diets in most developing countriesn. In this study, cowpea seeds were used to prepare cowpea four, protein isolates by the salt extractonndialysis method and peptdes by enzymatc hydrolysis using Alcalase and Flavourzymen Functonal analyses such as water absorpton capacity, oil absorpton capacity, emulsifying and foaming propertes were conducted on the cowpea peptdesn The physicochemical propertes determine their potental applicaton in food industries as functonal ingredientsn Cowpea peptdes could increase the value of cowpea by expanding its use, as well as contribute to the legume grain sector.

Keywords: physicochemical, peptides, Cowpea, alcalase, flavourzyme

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Authors: Rezvan Najafi, Korosh Heydari, Massoud Saidijam

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5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.

Keywords: colorectal cancer, miR-200c, 5-FU resistance, E-cadherin, PTEN

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Authors: Kalaumari Mayoral-Peña, Ana I. Montejano-Montelongo, Josué Reyes-Muñoz, Gonzalo A. Ortiz-Mancilla, Mayrin Rodríguez-Cruz, Víctor Hernández-Villalobos, Jesús A. Guzmán-López, Santiago García-Jacobo, Iván Licona-Vázquez, Grisel Fierros-Romero, Rosario Flores-Vallejo

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The group B-lactamic antibiotics include some of the most frequently used small drug molecules against bacterial infections. Nevertheless, an alarming decrease in their efficacy has been reported due to the emergence of antibiotic-resistant bacteria. Infections caused by bacteria expressing extended Spectrum B-lactamases (ESBLs) are difficult to treat and account for higher morbidity and mortality rates, delayed recovery, and high economic burden. According to the Global Report on Antimicrobial Resistance Surveillance, it is estimated that mortality due to resistant bacteria will ascend to 10 million cases per year worldwide. These facts highlight the importance of developing low-cost and readily accessible detection methods of drug-resistant ESBLs bacteria to prevent their spread and promote accurate and fast diagnosis. Bacterial detection is commonly done using molecular diagnostic techniques, where PCR stands out for its high performance. However, this technique requires specialized equipment not available everywhere, is time-consuming, and has a high cost. Loop-Mediated Isothermal Amplification (LAMP) is an alternative technique that works at a constant temperature, significantly decreasing the equipment cost. It yields double-stranded DNA of several lengths with repetitions of the target DNA sequence as a product. Although positive and negative results from LAMP can be discriminated by colorimetry, fluorescence, and turbidity, there is still a large room for improvement in the point-of-care implementation. DNA origami is a technique that allows the formation of 3D nanometric structures by folding a large single-stranded DNA (scaffold) into a determined shape with the help of short DNA sequences (staples), which hybridize with the scaffold. This research aimed to generate DNA origami structures using LAMP products as scaffolds to improve the sensitivity to detect ESBLs in point-of-care diagnosis. For this study, the coding sequence of the CTM-X-15 ESBL of E. coli was used to generate the LAMP products. The set of LAMP primers were designed using PrimerExplorerV5. As a result, a target sequence of 200 nucleotides from CTM-X-15 ESBL was obtained. Afterward, eight different DNA origami structures were designed using the target sequence in the SDCadnano and analyzed with CanDo to evaluate the stability of the 3D structures. The designs were constructed minimizing the total number of staples to reduce costs and complexity for point-of-care applications. After analyzing the DNA origami designs, two structures were selected. The first one was a zig-zag flat structure, while the second one was a wall-like shape. Given the sequence repetitions in the scaffold sequence, both were able to be assembled with only 6 different staples each one, ranging between 18 to 80 nucleotides. Simulations of both structures were performed using scaffolds of different sizes yielding stable structures in all the cases. The generation of the LAMP products were tested by colorimetry and electrophoresis. The formation of the DNA structures was analyzed using electrophoresis and colorimetry. The modeling of novel detection methods through bioinformatics tools allows reliable control and prediction of results. To our knowledge, this is the first study that uses LAMP products and DNA-origami in combination to delect ESBL-producing bacterial strains, which represent a promising methodology for diagnosis in the point-of-care.

Keywords: beta-lactamases, antibiotic resistance, DNA origami, isothermal amplification, LAMP technique, molecular diagnosis

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Authors: Zarmina Gillani, Mulazim Hussain Bukhari, Nuzhat Huma, Aqsa Qayyum

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Whey is a valuable by-product of dairy industry comprising of precious nutrients lactose, protein, vitamins and minerals for the human food but considered as a pollutant due to its biological activity. So, there is a need to develop nutritious whey products to overcome the problem of environmental pollution. This project was planned to develop a whey drink at different pasteurization temperatures and its quality was evaluated during storage. The result indicated that pH, acidity, total soluble solids and lactose content changed significantly (p < 0.01) due to lactic acid production during storage. Non-significant (p > 0.05) effects were detected on the protein and ash content of whey drink. Fat and viscosity changed significantly with respect to storage only. Sensory evaluation of whey drink revealed that both treatments remained acceptable while whey drink pasteurized at 75°C/30 minutes (WD2) gained more sensory score compared to whey drink pasteurized at 65°C/30minutes (WD1).

Keywords: pasteurization, sensory evaluation, storage, whey

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Authors: Viviane P. Romani, Bradley D. Olsen, Vilásia G. Martins

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Biodegradable films for food packaging have gained growing attention due to environmental pollution caused by synthetic films and the interest in the better use of resources from nature. Important research advances were made in the development of materials from proteins, polysaccharides, and lipids. However, the commercial use of these new generation of sustainable materials for food packaging is still limited due to their low mechanical and barrier properties that could compromise the food quality and safety. Thus, strategies to improve the performance of these materials have been tested, such as chemical modifications, incorporation of reinforcing structures and others. Cold plasma is a versatile, fast and environmentally friendly technology. It consists of a partially ionized gas containing free electrons, ions, and radicals and neutral particles able to react with polymers and start different reactions, leading to the polymer degradation, functionalization, etching and/or cross-linking. In the present study, biodegradable films from fish protein prepared through the casting technique were plasma treated using an AC glow discharge equipment. The reactor was preliminary evacuated to ~7 Pa and the films were exposed to air plasma for 2, 5 and 8 min. The films were evaluated by their mechanical and water vapor permeability (WVP) properties and changes in the protein structure were observed using Scanning Electron Microscopy (SEM) and X-ray diffraction (XRD). Potential cross-links and elimination of surface defects by etching might be the reason for the increase in tensile strength and decrease in the elongation at break observed. Among the times of plasma application tested, no differences were observed when higher times of exposure were used. The X-ray pattern showed a broad peak at 2θ = 19.51º that corresponds to the distance of 4.6Å by applying the Bragg’s law. This distance corresponds to the average backbone distance within the α-helix. Thus, the changes observed in the films might indicate that the helical configuration of fish protein was disturbed by plasma treatment. SEM images showed surface damage in the films with 5 and 8 min of plasma treatment, indicating that 2 min was the most adequate time of treatment. It was verified that plasma removes water from the films once weight loss of 4.45% was registered for films treated during 2 min. However, after 24 h in 50% of relative humidity, the water lost was recovered. WVP increased from 0.53 to 0.65 g.mm/h.m².kPa after plasma treatment during 2 min, that is desired for some foods applications which require water passage through the packaging. In general, the plasma technology affects the properties and structure of fish protein films. Since this technology changes the surface of polymers, these films might be used to develop multilayer materials, as well as to incorporate active substances in the surface to obtain active packaging.

Keywords: fish protein films, food packaging, improvement of properties, plasma treatment

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Authors: Keyur Raval, Steffen Krohn, Bruno Moerschbacher

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Chitin, the biopolymer of the N-acetylglucosamine, is the most abundant biopolymer on the planet after cellulose. Industrially, chitin is isolated and purified from the shell residues of shrimps. A deacetylated derivative of chitin i.e. chitosan has more market value and applications owing to it solubility and overall cationic charge compared to the parent polymer. This deacetylation on an industrial scale is performed chemically using alkalis like sodium hydroxide. This reaction not only is hazardous to the environment owing to negative impact on the marine ecosystem. A greener option to this process is the enzymatic process. In nature, the naïve chitin is converted to chitosan by chitin deacetylase (CDA). This enzymatic conversion on the industrial scale is however hampered by the crystallinity of chitin. Thus, this enzymatic action requires the substrate i.e. chitin to be soluble which is technically difficult and an energy consuming process. We in this project wanted to address this shortcoming of CDA. In lieu of this, we have modeled a fusion protein with CDA and an auxiliary protein. The main interest being to increase the accessibility of the enzyme towards crystalline chitin. A similar fusion work with chitinases had improved the catalytic ability towards insoluble chitin. In the first step, suitable partners were searched through the protein data bank (PDB) wherein the domain architecture were sought. The next step was to create the models of the fused product using various in silico techniques. The models were created by MODELLER and evaluated for properties such as the energy or the impairment of the binding sites. A fusion PCR has been designed based on the linker sequences generated by MODELLER and would be tested for its activity towards insoluble chitin.

Keywords: chitin deacetylase, modeling, chitin binding domain, chitinases

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Authors: Lali Shanshiashvili, Marika Chikviladze, Nino Mamulashvili, Maia Sepashvili, Nana Narmania, David Mikeladze

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Purpose: During demyelinating inflammatory diseases and after the damage of the myelin sheet, myelin-derived proteins, including myelin basic protein (MBP), are secreted into the extracellular space. MBP shows extensive post-translational modifications, including the deimination of arginine residues. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than the unmodified one. It is hypothesized that MBP could change the inflammatory response in astrocytes. Methods: MBP was isolated and purified from bovine brain white matter. Primary astrocyte cultures were prepared from whole brains of 2-day-old Wistar rats. For evaluation of glutamate uptake/release in astrocytes following treatment of cells with MBP charge isomers, Glutamate Assay Kit was used. The expression of EAAT-2 (excitatory amino acid transporters), peroxisome proliferator-activated receptor gamma (PPAR- γ), inhibitor of nuclear factor kappa B (IkB), and high mobility group protein B1 (HMGB1) in astrocytes were assayed by Western Blot analysis. Results: This study investigated the action of deiminated isomer (C8) on the cultured primary astrocytes and compared its effects with the effects of unmodified C1 isomers. The study found that C8 and C1 MBP differently act on the uptake and release of glutamate in astrocytes: nonmodified C1 MBP increases the uptake of glutamate and does not change the release, whereas C8 decreases the release of glutamate but does not alter the uptake. Nevertheless, both isomers increased the expression of PPAR-γ and EAAT2 in the same intensity. However, immunostaining and Western Blots of cell lysates showed a decrease of IkB and increased expression of HMGB1 after the treatment of astrocytes by C8. Moreover, in the presence of C8, astrocytes release more nitric oxide than unmodified C1 isomers. Conclusion: These data suggest that the deiminated isomer of MBP evokes an inflammatory response and enhances the ability of astrocytes to release proinflammatory mediators through activation of NF-kB after the breakdown of myelin sheets. Acknowledgment: This research was supported by the SRNSF Georgia RF17_534 grant.

Keywords: myelin basic protein, glutamate, deimination, astrocytes, inflammation

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Authors: Satoshi Furukawa, Satomu Morita, Lisa Wingenfeld, Katsuji Nishi, Masahito Hitosugi

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We studied the expression of hypoxia-related antigens (e.g., cold-inducible antigens and apoptotic antigens) in the myocardium and the cerebellumthat were obtained from individuals after death by carbon monoxide or hypothermia. The immunohistochemistry results revealed that expression of cold-inducible RNA binding protein (CIRBP) and RNA-binding protein 3 (RBM3) may be associated with hpyothermic and the hypoxic conditions. The expression of CIRBP and RBM3 in the myocardium was different from their expression in the cerebellum, especially in the Purkinje cells. The results indicate that agonal duration influences antigen expression. In the hypothermic condition, the myocardium uses more ATP since the force of the excitation-contraction coupling of the myocardium increases by more than 400% when the experimental temperature is reduced from 35°C to 25°C. The results obtained in this study indicate that physicians should pay attention to the myocardium when cooling the patient’s body to protect the brain.

Keywords: carbon monoxide death, cerebellum, CIRBP, hypothermic death, myocardium, RBM3

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Authors: Amena Omer Syeda, Harita Shyam

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Background: Anorexia a common symptom among patients with prolonged illness leading to anorexia-cachexia syndrome with a prevalence rate of 70%. In order to provide effective health care and better response to treatment, appetite should be assessed on admission and then periodically for earlier nutrition intervention. Functional Assessment of Anorexia/Cachexia Therapy (FAACT) appetite scale is 12 questions, patient-rated, symptom specific measure for appetite, and distress from anorexia. It assigns a score ranging from 0 (worst response) to 4 (best response). Therefore, proposing a total score of ≤24 may be sufficient to make a diagnosis of anorexia. Objectives: To assess the FAACT scale by co-relating the scores with the Nutritional intake and BMI of Cancer Patients. Methods: The FAACT scores of 100 cancer in-patients receiving chemotherapy or radiation as treatment, their 24-hour calorie and protein intake and BMI were recorded. The data was then statistically analyzed. Results: The calorie and protein intake and FAACT scores both showed a significant positive co-relation (p<0.001), inferring that the patients with a FAACT score of ≤24 where not meeting their calorie as well as protein requirements, hence rightly categorizing them as anorexic. The co-relation between BMI and FAACT scores showed a weak co-relation and was not statistically significant (p > 0.05).The FAACT scale thus is not sensitive to distinguish patients being under-weight, normal weight or obese. Conclusion: The FAACT scale helps in providing better palliative and nutritional care as it correctly assessed anorexia /cachexia in cancer patients and co-related significantly with their nutrient intake.

Keywords: appetite, cachexia, cancer, malnutrition

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Authors: Wenfa Yu, Thuva Gnanasampanthan, John Finlay, Jessica Clarke, Charlotte Anderson, Tony Clare, Axel Rosenhahn

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Marine biofouling is a worldwide problem at vast economic and ecological costs. Historically it was combated with toxic coatings such as tributyltin. As those coatings being banned nowadays, finding environmental friendly antifouling solution has become an urgent topic. In this study antifouling coatings consisted of natural occurring polysaccharides hyaluronic acid (HA), alginic acid (AA), chitosan (Ch) and polyelectrolyte polyethylenimine (PEI) are constructed into polyelectrolyte multilayers (PEMs) in a Layer-by-Layer (LbL) method. LbL PEM construction is a straightforward way to assemble biomacromolecular coatings on surfaces. Advantages about PEM include ease of handling, highly diverse PEM composition, precise control over the thickness and so on. PEMs have been widely employed in medical application and there are numerous studies regarding their protein adsorption, elasticity and cell adhesive properties. With the adjustment of coating composition, termination layer charge, coating morphology and cross-linking method, it is possible to prepare low marine biofouling coatings with PEMs. In this study, using spin coating technology, PEM construction was achieved at smooth multilayers with roughness as low as 2nm rms and highly reproducible thickness around 50nm. To obtain stability in sea water, the multilayers were covalently cross-linked either thermally or chemically. The cross-linking method affected surface energy, which was reflected in water contact angle, thermal cross-linking led to hydrophobic surfaces and chemical cross-linking generated hydrophilic surfaces. The coatings were then evaluated regarding its protein resistance and biological species resistance. While the hydrophobic thermally cross-linked PEM had low resistance towards proteins, the resistance of chemically cross-linked PEM strongly depended on the PEM termination layer and the charge of the protein, opposite charge caused high adsorption and same charge low adsorption, indicating electrostatic interaction plays a crucial role in the protein adsorption processes. Ulva linza was chosen as the biological species for antifouling performance evaluation. Despite of the poor resistance towards protein adsorption, thermally cross-linked PEM showed good resistance against Ulva spores settlement, the chemically cross-linked multilayers showed poor resistance regardless of the termination layer. Marine species adhesion is a complex process, although it involves proteins as bioadhesives, protein resistance its own is not a fully indicator for its antifouling performance. The species will pre select the surface, responding to cues like surface energy, chemistry, or charge and so on. Thus making it difficult for one single factors to determine its antifouling performance. Preparing PEM coating is a comprehensive work involving choosing polyelectrolyte combination, determining termination layer and the method for cross-linking. These decisions will affect PEM properties such as surface energy, charge, which is crucial, since biofouling is a process responding to surface properties in a highly sensitive and dynamic way.

Keywords: hyaluronic acid, polyelectrolyte multilayers, protein resistance, Ulva linza zoospores

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Authors: Chang Liang, Weizhi Gong, Yan Zhang

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Purpose: Hepatocellular carcinoma (HCC) is a malignant tumor with a high mortality rate and poor prognosis worldwide. Murine double minute 2 (MDM2) regulates the tumor suppressor p53, increasing cancer risk and accelerating tumor progression. Speckle-type POX virus and zinc finger protein (SPOP), a key of subunit of Cullin-Ring E3 ligase, inhibits tumor genesis and progression by the ubiquitination of its downstream substrates. This study aimed to clarify whether SPOP and MDM2 are mutually regulated in HCC and the correlation between SPOP and MDM2 and the prognosis of HCC patients. Methods: First, the expression of SPOP and MDM2 in HCC tissues were detected by TCGA database. Then, 53 paired samples of HCC tumor and adjacent tissues were collected to evaluate the expression of SPOP and MDM2 using immunohistochemistry. Chi-square test or Fisher’s exact test were used to analyze the relationship between clinicopathological features and the expression levels of SPOP and MDM2. In addition, Kaplan‒Meier curve analysis and log-rank test were used to investigate the effects of SPOP and MDM2 on the survival of HCC patients. Last, the Multivariate Cox proportional risk regression model analyzed whether the different expression levels of SPOP and MDM2 were independent risk factors for the prognosis of HCC patients. Results: Bioinformatics analysis revealed the low expression of SPOP and high expression of MDM2 were related to worse prognosis of HCC patients. The relationship between the expression of SPOP and MDM2 and tumor stem-like features showed an opposite trend. The immunohistochemistry showed the expression of SPOP protein was significantly downregulated while MDM2 protein significantly upregulated in HCC tissue compared to that in para-cancerous tissue. Tumors with low SPOP expression were related to worse T stage and Barcelona Clinic Liver Cancer (BCLC) stage, but tumors with high MDM2 expression were related to worse T stage, M stage, and BCLC stage. Kaplan–Meier curves showed HCC patients with high SPOP expression and low MDM2 expression had better survival than those with low SPOP expression and high MDM2 expression (P < 0.05). A multivariate Cox proportional risk regression model confirmed that a high MDM2 expression level was an independent risk factor for poor prognosis in HCC patients (P <0.05). Conclusion: The expression of SPOP protein was significantly downregulated, while the expression of MDM2 significantly upregulated in HCC. The low expression of SPOP and high expression. of MDM2 were associated with malignant progression and poor prognosis of HCC patients, indicating a potential therapeutic target for HCC patients.

Keywords: hepatocellular carcinoma, murine double minute 2, speckle-type POX virus and zinc finger protein, ubiquitination

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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: 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: Lucretia Ramnath

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Fishmeal is extensively used for fish farming but is an expensive fish feed ingredient. A cheaper alternate to fishmeal is single cell protein (SCP) which can be cultivated on fermentable sugars recovered from organic waste streams such as pulp and paper mill sludge (PPMS). PPMS has a high cellulose content, thus is suitable for glucose recovery through enzymatic hydrolysis but is hampered by lignin and ash. To render PPMS amenable for enzymatic hydrolysis, the PPMS waspre-treated to produce a glucose-rich hydrolysate which served as a feed stock for the production of fungal SCP. The PPMS used in this study had the following composition: 72.77% carbohydrates, 8.6% lignin, and 18.63% ash. The pre-treatments had no significant effect on lignin composition but had a substantial effect on carbohydrate and ash content. Enzymatic hydrolysis of screened PPMS was previously optimized through response surface methodology (RSM) and 2-factorial design. The optimized protocol resulted in a hydrolysate containing 46.1 g/L of glucose, of which 86% was recovered after downstream processing by passing through a 100-mesh sieve (38 µm pore size). Vogel’s medium supplemented with 10 g/L hydrolysate successfully supported the growth of Fusarium venenatum, conducted using standard growth conditions; pH 6, 200 rpm, 2.88 g/L ammonium phosphate, 25°C. A maximum F. venenatum biomass of 45 g/L was produced with a yield coefficient of 4.67. Pulp and paper mill sludge hydrolysate contained approximately five times more glucose than what was needed for SCP production and served as a suitable carbon source. We have shown that PPMS can be successfully beneficiated for SCP production.

Keywords: pulp and paper waste, fungi, single cell protein, hydrolysate

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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: 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: Krishna Pal Singh, Shailendra Kumar Gupta, Olaf Wolkenhauer

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Background: Our study aimed to identify common genes and potential targets across the four diseases, which include rheumatoid arthritis, melanoma metastasis, ulcerative colitis, and Crohn’s disease. We used a network and systems biology approach to identify the hub gene, which can act as a potential target for all four disease conditions. The regulatory network was extracted from the PPI using the MCODE module present in Cytoscape. Our objective was to investigate the significance of hub genes in these diseases using gene ontology and KEGG pathway enrichment analysis. Methods: Our methodology involved collecting disease gene-related information from DisGeNET databases and performing protein-protein interaction (PPI) network and core genes screening. We then conducted gene ontology and KEGG pathway enrichment analysis. Results: We found that IL6 plays a critical role in all disease conditions and in different pathways that can be associated with the development of all four diseases. Conclusions: The theoretical importance of our research is that we employed various systems and structural biology techniques to identify a crucial protein that could serve as a promising target for treating multiple diseases. Our data collection and analysis procedures involved rigorous scrutiny, ensuring high-quality results. Our conclusion is that IL6 plays a significant role in all four diseases, and it can act as a potential target for treating them. Our findings may have important implications for the development of novel therapeutic interventions for these diseases.

Keywords: melanoma metastasis, rheumatoid arthritis, inflammatory bowel diseases, integrated bioinformatics analysis

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Authors: Pedro G. Morouço

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One utmost challenge in Tissue Engineering is the production of 3D constructs capable of mimicking the functional hierarchy of native tissues. This is well stated for osteochondral tissue due to the complex mechanical functional unit based on the junction of articular cartilage and bone. Thus, the aim of the present study was to develop a new additive manufacturing system coupling micro-extrusion with hydrogels printing. An integrated system was developed with 2 main features: (i) the printing of up to three distinct hydrogels; (ii) in coordination with the printing of a thermoplastic structural support. The hydrogel printing module was projected with a ‘revolver-like’ system, where the hydrogel selection was made by a rotating mechanism. The hydrogel deposition was then controlled by pressured air input. The use of specific components approved for medical use was incorporated in the material dispensing system (Nordson EDF Optimum® fluid dispensing system). The thermoplastic extrusion modulus enabled the control of required extrusion temperature through electric resistances in the polymer reservoir and the extrusion system. After testing and upgrades, a hydrogel modulus with 3 syringes (3cm3 capacity each), with a pressure range of 0-2.5bar, a rotational speed of 0-5rpm, and working with needles from 200-800µm was obtained. This modulus was successfully coupled to the extrusion system that presented a temperature up to 300˚C, a pressure range of 0-12bar, and working with nozzles from 200-500µm. The applied motor could provide a velocity range 0-2000mm/min. Although, there are distinct printing requirements for hydrogels and polymers, the novel system could develop hybrid scaffolds, combining the 2 moduli. The morphological analysis showed high reliability (n=5) between the theoretical and obtained filament and pore size (350µm and 300µm vs. 342±4µm and 302±3µm, p>0.05, respectively) of the polymer; and multi-material 3D constructs were successfully obtained. Human tissues present very distinct and complex structures regarding their mechanical properties, organization, composition and dimensions. For osteochondral regenerative medicine, a multiphasic scaffold is required as subchondral bone and overlying cartilage must regenerate at the same time. Thus, a scaffold with 3 layers (bone, intermediate and cartilage parts) can be a promising approach. The developed system may give a suitable solution to construct those hybrid scaffolds with enhanced properties. The present novel system is a step-forward regarding osteochondral tissue engineering due to its ability to generate layered mechanically stable implants through the double-printing of hydrogels with thermoplastics.

Keywords: 3D bioprinting, bone regeneration, cartilage regeneration, regenerative medicine, tissue engineering

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Authors: Dinara Ikramova, Karin A. Hing, Simon C. F. Rawlinson

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Silicate-substituted hydroxyapatite (SiHA) has been shown to enhance bone regeneration in vivo compared with phase pure stoichiometric hydroxyapatite. Evidence suggests that substrate chemistry dependent formation of a permissive protein layer on the surface of synthetic bone graft substitute materials is key for bioactivity and cell attachment. However, little information is available on whether the substrate chemistry may affect cell migration and recruitment. The aim of this study is to investigate whether or not human Mesenchymal Stem Cells (hMSCs) exhibit a chemotactic response to SiHA porous granules and if it can be linked to either the ion exchange or protein sequestering and enrichment on the surface of the material. 150mg of SiHA granules with 80% total porosity and 20% strut porosity were incubated in 1ml of either Serum Free Media (SFM) or 10% Serum Containing Media (SCM) under static cell culture conditions (37°C, 5% CO2) in absence of cells. Protein sequestering and exchange of calcium, phosphate and silicate ions were analysed at 0.5, 1, 2, 4, 8, 16 and 24 hours with n=12 per time point. Migration of hMSCs in the presence of 150mg of SiHA granules was assessed over 24 hours using a modified transwell migration system in either SFM or SCM (n=6) with 30% serum containing media acting as a positive control. At 24 hours protein sequestering and ionic exchange were analysed, and the number of cells was quantified using a high throughput confocal microscope (IN Cell Analyser 6000). In acellular condition, both calcium and phosphate ion concentrations in media showed a decrease at 24 hours which was greater in SFM than in SCM. This suggests possible formation and precipitation of a bone like apatite on the surface of SiHA. Reduction in this activity observed in SCM indicates that the presence of serum proteins is interfering with the ion exchange at the material and media interface. Adsorbed protein levels showed fluctuation over time followed by sharp decrease at 24 hours, suggesting a possible protein rearrangement on the surface of the material. The ion analysis performed on SFM and SCM after 24-hour incubation with cells in the presence of granules showed a greater reduction in phosphate concentration in both SFM and SCM compared to phosphate levels in acellular condition. Silicate concentration in SCM increased from 1.6mM (absence of cells) to 5.1mM (presence of cells). This indicates that the cells are promoting the uptake of phosphate and release of silicate ions. No significant change was seen in levels of adsorbed proteins in the presence and absence of cells. Further analysis is required to determine whether the species of these proteins change over time. The analysis of cell migration after 24-hour incubation showed more cells migrating towards the granules, 12.7% in SFM and 8.3% in SCM, than in positive control, 4.5% in SFM and 3.6% in SCM respectively. These results suggest that SiHA has a chemotactic activity independent of serum proteins. A property which has not previously been demonstrated for a synthetic bone graft material.

Keywords: cell migration, hMSCs, SiHA, transwell migration system

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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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