Search results for: G protein α subunit

Authors: Isobel Hambleton, Mark Carrington

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Trypanosoma brucei, the causal agent of a range of diseases in humans and livestock, evades the mammalian immune system through a population survival strategy based on the expression of a series of antigenically distinct variant surface glycoproteins (VSGs). RNAi mediated knockdown of the active VSG gene triggers a precytokinesis cell cycle arrest. To determine whether this phenotype is the result of reduced VSG transcript or depleted VSG protein, we used morpholino antisense oligonucleotides to block translation of VSG mRNA. The same precytokinesis cell cycle arrest was observed, suggesting that VSG protein abundance is monitored closely throughout the cell cycle. An inducible expression system has been developed to test various GPI-anchored proteins for their ability to rescue this cell cycle arrest. This system has been used to demonstrate that wild-type VSG expressed from a T7 promoter rescues this phenotype. This indicates that VSG expression from one of the specialised bloodstream expression sites (BES) is not essential for cell division. The same approach has been used to define the minimum essential features of a VSG necessary for function.

Keywords: bloodstream expression site, morpholino, precytokinesis cell cycle arrest, variant surface glycoprotein

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Authors: Elene Zhuravliova, Tamar Barbakadze, Irina Kalandadze, Elnari Zaalishvili, Lali Shanshiashvili, David Mikeladze

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Background: Multiple sclerosis (MS) is an inflammatory, neurodegenerative disease, which is accompanied by demyelination and autoimmune response to myelin proteins. Among post-translational modifications, which mediate the modulation of inflammatory pathways during MS, methylation is the main one. The methylation of DNA, also amino acids lysine and arginine, occurs in the cell. It was found that decreased trans-methylation is associated with neuroinflammatory diseases. Therefore, abnormal regulation of the methyl cycle could induce demyelination through the action on PAD (peptidyl-arginine-deiminase) gene promoter. PAD takes part in protein citrullination and targets myelin basic protein (MBP), which is affected during demyelination. To determine whether MBP charge isomers are changing the methyl cycle, we have estimated the concentrations of methyl cycle metabolites in MBP-activated primary astrocytes and oligodendrocytes. For this purpose, the action of the citrullinated MBP- C8 and the most cationic MBP-C1 isomers on the primary cells were investigated. Methods: Primary oligodendrocyte and astrocyte cell cultures were prepared from whole brains of 2-day-old Wistar rats. The methyl cycle metabolites, including homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH), were estimated by HPLC analysis using fluorescence detection and prior derivatization. Results: We found that the action of MBP-C8 and MBP-C1 induces a decrease in the concentration of both methyl cycle metabolites, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), in astrocytes compared to the control cells. As for oligodendrocytes, the concentration of SAM was increased by the addition of MBP-C1, while MBP-C8 has no significant effect. As for SAH, its concentration was increased compared to the control cells by the action of both MBP-C1 and MBP-C8. A significant increase in homocysteine concentration was observed by the action of the MBP-C8 isomer in both oligodendrocytes and astrocytes. Conclusion: These data suggest that MBP charge isomers change the concentration of methyl cycle metabolites. MBP-C8 citrullinated isomer causes elevation of homocysteine in astrocytes and oligodendrocytes, which may be the reason for decreased astrocyte proliferation and increased oligodendrocyte cell death which takes place in neurodegenerative processes. Elevated homocysteine levels and subsequent abnormal regulation of methyl cycles in oligodendrocytes possibly change the methylation of DNA that activates PAD gene promoter and induces the synthesis of PAD, which in turn provokes the process of citrullination, which is the accompanying process of demyelination. Acknowledgment: This research was supported by the SRNSF Georgia RF17_534 grant.

Keywords: myelin basic protein, astrocytes, methyl cycle metabolites, homocysteine, oligodendrocytes

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Authors: Usman Mir Khan, Ishtiaque Ahmad, Saima Inayat, H. M. Arslan Amin, Muhammad Ayaz, Nisar Ahmad

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Citrus reticulata Blanco crude flowers extract (CFE) at four different concentration (1, 2, 3 and 4%, v/v) were used as natural milk coagulant instead of rennet to apply for Cheddar cheese making from buffalo milk. The physicochemical properties and nutrition composition of Cheddar cheeses were compared with cheese made with 0.002% (v/v) rennet (control cheese). Physico-chemical of Cheddar cheese showed that cheese made with 1% and 2% of CFE had a crumbly and slightly softer texture of cheese. While, cheeses containing 3 and 4% CFE had semi-hard textural properties of curd similar to rennet added cheese. The CFE made cheese had moisture 37 %, fat 45 % on dry basis similar to rennet made Cheddar cheese. Protein analysis shows that CFE made cheese had significant higher protein content than control. The Cheddar cheese with 3% and 1% CFE were preferred by consumers instead of 2% and 4% CFE for their taste, texture/appearance and overall acceptability. Conclusively, CFE coagulated Cheddar cheese fulfills the nutritional requirement with acceptable organoleptic characteristics and at the same time provides nutritional health benefits.

Keywords: cheddar cheese, Citrus reticulata Blanco, buffalo milk, milk coagulant

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Authors: Akram Abi, Clarissa Müller, Hans-Joachim Jördening

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Laminaribiose is a beta-1,3-glycoside which is used in the medical field for the treatment of dermatitis and also can be used as a building block for new pharmaceutics. The conventional process of laminaribiose production is the uneconomical process of hydrolysis of laminarin extracted from natural polysaccharides of plant origin. A more economical approach however is attainable by enzymatically synthesis of laminaribiose via a reverse phosphorylase reaction catalyzed by laminaribiose phosphorylase (LP) from Euglena gracilis. Different cultivation methods of Euglena gracilis and the effect on LP production have been investigated. Buffered/unbuffered heterotrophic and mixotrophic cultivations of Euglena gracilis has been carried out. Changes of biomass and LP production, glucose level and pH, cell count and shape has been monitored in the course of time. The results obtained from experiments each in three repetitions, show that in the heterotrophic cultivation of Euglena gracilis not only more biomass is produced compared to mixotrophic cultivation, but also higher specific protein concentration is achieved. Furthermore, the LP activity test showed that the protein extracted from heterotrophically cultured cells has a higher LP activity. It was also observed that the cells develop in a distinctive different shape between these two cultures and have different length to width ratios. Taking the heterotrophic culture as the more efficient cultivation method in LP production, another comparative experiment between buffered and unbuffered heterothrophic culture was carried out that showed the unbuffered culture has advantages over the other one in respect of both LP production and resulting activity. A hetrotrophic cultivation of Euglena gracilis in a 5L bioreactor with controlled operating conditions showed a distinctive improvement of all the aspects of culture compared to the shaking flask cultivations. Biomass production was improved from 5 to more than 8 g/l (dry weight) which resulted in a specific protein concentration of 45 g/l in the heterotrophic cultivation in the bioreactor. In further attempts to improve LP production, different purification methods were tested and each method was checks through an activity assay. A laminaribiose yield of 35% was achieved which was by far the highest amount amongst different methods tested.

Keywords: euglena gracilis, heterotrophic culture, laminaribiose production, mixotrophic culture

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Authors: Kerekoppa Puttaiah Bhatta Ramesha, Uday Kannegundla, Praseeda Mol, Lathika Gopalakrishnan, Jagish Kour Reen, Gourav Dey, Manish Kumar, Sakthivel Jeyakumar, Arumugam Kumaresan, Kiran Kumar M., Thottethodi Subrahmanya Keshava Prasad

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Spermatozoa are the highly specialized transcriptionally and translationally inactive haploid male gamete. The understanding of proteome of sperm is indispensable to explore the mechanism of sperm motility and fertility. Though there is a large number of human sperm proteomic studies, in-depth proteomic information on Bos indicus spermatozoa is not well established yet. Therefore, we illustrated the profile of sperm proteome in indigenous cattle, Malnad gidda (Bos Indicus), using high-resolution mass spectrometry. In the current study, two semen ejaculates from 3 breeding bulls were collected employing the artificial vaginal method. Using 45% percoll purification, spermatozoa cells were isolated. Protein was extracted using lysis buffer containing 2% Sodium Dodecyl Sulphate (SDS) and protein concentration was estimated. Fifty micrograms of protein from each individual were pooled for further downstream processing. Pooled sample was fractionated using SDS-Poly Acrylamide Gel Electrophoresis, which is followed by in-gel digestion. The peptides were subjected to C18 Stage Tip clean-up and analyzed in Orbitrap Fusion Tribrid mass spectrometer interfaced with Proxeon Easy-nano LC II system (Thermo Scientific, Bremen, Germany). We identified a total of 6773 peptides with 28426 peptide spectral matches, which belonged to 1081 proteins. Gene ontology analysis has been carried out to determine the biological processes, molecular functions and cellular components associated with sperm protein. The biological process chiefly represented our data is an oxidation-reduction process (5%), spermatogenesis (2.5%) and spermatid development (1.4%). The highlighted molecular functions are ATP, and GTP binding (14%) and the prominent cellular components most observed in our data were nuclear membrane (1.5%), acrosomal vesicle (1.4%), and motile cilium (1.3%). Seventeen percent of sperm proteins identified in this study were involved in metabolic pathways. To the best of our knowledge, this data represents the first total sperm proteome from indigenous cattle, Malnad Gidda. We believe that our preliminary findings could provide a strong base for the future understanding of bovine sperm proteomics.

Keywords: Bos indicus, Malnad Gidda, mass spectrometry, spermatozoa

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Authors: Ashima Sharma, Tapan K. Chaudhuri

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Human Serum Albumin (HSA) is one of the most demanded therapeutic protein with immense biotechnological applications. The current source of HSA is human blood plasma. Blood is a limited and an unsafe source as it possesses the risk of contamination by various blood derived pathogens. This issue led to exploitation of various hosts with the aim to obtain an alternative source for the production of the rHSA. But, till now no host has been proven to be effective commercially for rHSA production because of their respective limitations. Thus, there exists an indispensable need to promote non-animal derived rHSA production. Of all the host systems, Escherichia coli is one of the most convenient hosts which has contributed in the production of more than 30% of the FDA approved recombinant pharmaceuticals. E. coli grows rapidly and its culture reaches high cell density using inexpensive and simple substrates. The fermentation batch turnaround number for E. coli culture is 300 per year, which is far greater than any of the host systems available. Therefore, E. coli derived recombinant products have more economical potential as fermentation processes are cheaper compared to the other expression hosts available. Despite of all the mentioned advantages, E. coli had not been successfully adopted as a host for rHSA production. The major bottleneck in exploiting E. coli as a host for rHSA production was aggregation i.e. majority of the expressed recombinant protein was forming inclusion bodies (more than 90% of the total expressed rHSA) in the E. coli cytosol. Recovery of functional rHSA form inclusion body is not preferred because it is tedious, time consuming, laborious and expensive. Because of this limitation, E. coli host system was neglected for rHSA production for last few decades. Considering the advantages of E. coli as a host, the present work has targeted E. coli as an alternate host for rHSA production through resolving the major issue of inclusion body formation associated with it. In the present study, we have developed a novel and innovative method for enhanced soluble and functional production of rHSA in E.coli (~60% of the total expressed rHSA in the soluble fraction) through modulation of the cellular growth, folding and environmental parameters, thereby leading to significantly improved and enhanced -expression levels as well as the functional and soluble proportion of the total expressed rHSA in the cytosolic fraction of the host. Therefore, in the present case we have filled in the gap in the literature, by exploiting the most well studied host system Escherichia coli which is of low cost, fast growing, scalable and ‘yet neglected’, for the enhancement of functional production of HSA- one of the most crucial biomolecule for clinical and biotechnological applications.

Keywords: enhanced functional production of rHSA in E. coli, recombinant human serum albumin, recombinant protein expression, recombinant protein processing

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Authors: Marzieh Eshaghi Koupaei

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By cultivating mealworm, we reduce greenhouse gases and avoid the use of transgenic products such as soybeans, and we provide food resources rich in protein, amino acids, minerals, etc. for humans and animals, and it has created employment and entrepreneurship. We serve the environment by producing oil from mealworm in the cosmetic industry, using its waste as organic fertilizer and its powder in bodybuilding, and by breaking down plastic by mealworm. The production and breeding of mealworm requires very little infrastructure and does not require much trouble, and requires very little food, and reproduces easily and quickly, and a mealworm production workshop is noiseless, odorless, and pollution-free And the costs are very low. It is possible to use third grade fruits and unsalable fruits of farmers to feed the mealworms, which is completely economical and cost-effective. Mealworms can break down plastic in their intestines and turn it into carbon dioxide. . This process was done in only 16 days, which is a very short time compared to several centuries for plastic to decompose. By producing mealworm, we have helped to preserve the environment and provided the source of protein needed by humans and animals. This industrial insect has the ability and value of commercialization and creates employment and helps the economy of the society.

Keywords: breeding, production of insects, mealworms, research, animal feed, human feed

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Authors: Jyh-Cheng Chen, Tai-Jing Wang, Yun-Wei Lin

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Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Treatment with astaxanthin decreased Rad51 expression and phospho-AKT protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector significantly rescued the decreased Rad51 protein and mRNA levels in astaxanthin-treated NSCLC cells. Combined treatment with PI3K inhibitors (LY294002 or wortmannin) and astaxanthin further decreased the Rad51 expression in NSCLC cells. Knockdown of Rad51 enhanced astaxanthin-induced cytotoxicity and growth inhibition in NSCLC cells. These findings may have implications for the rational design of future drug regimens incorporating astaxanthin for the treatment of NSCLC.

Keywords: astaxanthin, cytotoxicity, AKT, non-small cell lung cancer, PI3K

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Authors: Muhammad Zubair, Aman Ullah, Jianping Wu

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During the last decades, petroleum derived synthetic polymers like polyethylene terephthalate, polyvinylchloride, polyethylene, polypropylene and polystyrene has extensively been used in the field of food packaging and mostly are non-degradable. Biopolymers are a good fit for single-use or short-lived products such as food packaging. Spent hens, a poultry by-product which is of little economic value and their disposal are environmentally harmful. Through current study, we have explored the possibility to transform proteins from spent fowl into green food packaging material. Proteins from spent fowl were extracted within 1 hour using pH shift method with recovery of about 74%. Different plasticizers were tried like glycerol, sorbitol, glutaraldehyde, 1,2 ethylene glycol and 1,2 butanediol. Glycerol was the best plasticizer among all these plasticizers. A naturally occurring and non-toxic cross-linking agent, chitosan, was used to form the chitosan/glycerol/protein blend by casting and compression molding techniques. The mechanical properties were characterized using tensile strength analyzer. The nano-reinforcements with homogeneous dispersion of nanoparticles lead to improved physical properties suggesting that these materials have great potential for food packaging applications.

Keywords: differential scanning calorimetry, dynamic mechanical analysis, scanning electron microscopy, spent hen

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Authors: Sajjad ur Rahman, Mariam Azam, Mukarram Bashir, Seemal Javaid, Aoun Muhammad, Muhammad Tahir, Jawad, Hannan Khan, Muhammad Zohaib

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Partially fermented soya hulls and wheat bran through Saccharomyces cerevisiae (DL-22 S/N) substantiated as a natural source for quality milk production. Saccharomyces cerevisiae (DL-22 S/N) were grown under in-vivo conditions and processed through two-step fermentation with substrates. The extra pure metabolites (XPM) were dried and processed for maintaining 1mm mesh size particles for supplementation of pelleted feed. Two groups of a cow (Holstein Friesian) having 8 animals of similar age and lactation were given the experimental concentrates. Group A was fed daily with 12gm of XPM and 22% protein-pelleted feed, while Group B was provided with no metabolites in their feed. In thirty-nine days of trial, improvement in the overall health, body score, milk protein, milk fat, ash, and solid not fat (SNF), yield, and incidence rate of mastitis was observed. The collected data revealed an improvement in milk production of 2.02 liter/h/d. However, a reduction (3.75%) in the milk fats and an increase in the milk SNF was around 0.58%. The ash content ranged between 6.4-7.5%. The incidence of mastitis was reduced to less than 2%.

Keywords: microbial metabolites, Saccharomyces cerevisiae, milk production, fermentation, post-biotic metabolites, immunity

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Authors: Vita Sterna, Sanita Zute, Inga Jansone, Linda Brunava, Inara Kantane

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Grains, including oats (Avena sativa L.), have been recognized functional foods, because provide beneficial effect on the health of the consumer and decrease the risk of various diseases.Oats are good source of soluble fibre, essential amino acids, unsaturated fatty acids, vitamins and minerals. Oat breeders have developed oat varieties and improved yielding ability potential of oat varieties. Therefore, the aim of investigation was to analyze the composition of perspective oat varieties and breeding lines grains grown in different conditions and evaluate functional properties. In the studied samples content of protein, starch, β - glucans, total dietetic fibre, composition of amino acids and vitamin E were determined. The results of analysis showed that protein content depending of varieties ranged 9.70 –17.30% total dietary fibre 13.66-30.17 g100g-1, content of β-glucans 2.7-3.5 g100g-1, amount of vitamin E (α-tocopherol) determined from 4 to 9.9 mg kg-1. The sum of essential amino acids in oat grain samples were determined from 31.63 to 54.90 gkg-1. Concluded that amino acids composition of husked and naked oats grown in organic or conventional conditions is close to optimal.

Keywords: dietetic fibre, amino acids, scores, nutrition value

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Authors: Soledad Carinelli, Iñigo Fernández, José Luis González-Mora, Pedro A. Salazar-Carballo

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Mastitis is the most relevant inflammatory disease in cattle, affecting the animal health and causing important economic losses on dairy farms. This disease takes place in the mammary gland or udder when some opportunistic microorganisms, such as Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium bovis, etc., invade the teat canal. According to the severity of the inflammation, mastitis can be classified as sub-clinical, clinical and chronic. Standard methods for mastitis detection include counts of somatic cells, cell culture, electrical conductivity of the milk, and California test (evaluation of “gel-like” matrix consistency after cell lysed with detergents). However, these assays present some limitations for accurate detection of subclinical mastitis. Currently, haptoglobin, an acute phase protein, has been proposed as novel and effective biomarker for mastitis detection. In this work, an electrochemical biosensor based on polydopamine-modified magnetic nanoparticles (MNPs@pDA) for haptoglobin detection is reported. Thus, MNPs@pDA has been synthesized by our group and functionalized with hemoglobin due to its high affinity to haptoglobin protein. The protein was labeled with specific antibodies modified with alkaline phosphatase enzyme for its electrochemical detection using an electroactive substrate (1-naphthyl phosphate) by differential pulse voltammetry. After the optimization of assay parameters, the haptoglobin determination was evaluated in milk. The strategy presented in this work shows a wide range of detection, achieving a limit of detection of 43 ng/mL. The accuracy of the strategy was determined by recovery assays, being of 84 and 94.5% for two Hp levels around the cut off value. Milk real samples were tested and the prediction capacity of the electrochemical biosensor was compared with a Haptoglobin commercial ELISA kit. The performance of the assay has demonstrated this strategy is an excellent and real alternative as screen method for sub-clinical bovine mastitis detection.

Keywords: bovine mastitis, haptoglobin, electrochemistry, magnetic nanoparticles, polydopamine

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Authors: Farid Shekari

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Salicylic Acid (SA) is a plant hormone that improves some physiological responses of plants under stress conditions. Seeds of two desi type chickpea cultivars, viz., Kaka and Pirooz, primed with 250, 500, 750, and 1000 μM of SA and a group of seeds without any treating (as control) were evaluated under rain fed conditions. Seed priming in both cultivars led to higher efficiency compare to non-primed treatments. In general, seed priming with 500 and 750 μM of SA had appropriate effects; however the cultivars responses were different in this regard. Kaka showed better performance both in primed and non-primed seed than Pirooz. Results of this study revealed that not only yield quantity but also yield quality, as seed protein amounts, could positively affect by SA treatments. It seems that SA by enhancing of soluble sugars and proline amounts enhanced total water potential (ψ) and RWC. The increment in RWC led to rose of chlorophyll content of plants chlorophyll stability. In general, SA increased water use efficiency, both in biologic and seed yield base, and drought tolerance of chickpea plants. HI was a little decreased in SA treatments and it shows that SA more effective in biomass production than seed yield.

Keywords: chlorophyll, harvest index, proline, seed protein, soluble sugar, water use efficiency, yield component

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Authors: Hasan Alp Sahin, Ergin Ozturk

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The effects of raw bee propolis (RP) and water (WEP) or ethanol (EEP) extract of propolis on growth performance, selected immune parameters (IgA, IgY and IgM) and some blood parameters such as aspartate aminotransferase, alanine aminotransferase, trygliceride, total protein, albumin, calcium, phosphorus, total antioxidant status and total oxidant status were determined. The study was conducted between 15th and 20th weeks (6 weeks) and used a total of 48 broiler breeder pullets (Ross-308). The broiler breeder in control group was fed diet without propolis whereas the birds in RP, WEP and EEP groups were fed diets with RP, WEP and EEP at the level of 1200, 400 and 400 ppm, respectively. All pullets were fed mash form diet with 15% crude protein and 2800 ME kcal/kg. All propolis forms had not a beneficial effect on any studied parameters compared to control group (P > 0.05). The results of the study indicated that both the level of the active matters supplied from the bee propolis has no enough beneficial effect on performance, some immune and blood parameters on broiler breeders or they did not have such a level that would cause a beneficial effect on these variables.

Keywords: antioxidant, bee product , poultry breeders, growth performance, immune parameters, blood chemistry

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Authors: A. T. Ramachandra Naik, H. Shivananda Murthy, H. n. Anjanayappa

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The freshwater prawn, Macrobrachium rosenbergii is a more popular crustacean cultured widely in monoculture system in India. It has got high nutritional value in the human diet. Hence, understanding its enzymatic and body composition is important in order to judge its flesh quality. Fish oil specially derived from Indian oil sardine is a good source of highly unsaturated fatty acid and lipid source in fish/prawn diet. A 35% crude protein diet with graded levels of Sardine oil as a source of fat was incorporated at four levels viz, 2.07, 4.07, 6.07 and 8.07% maintaining a total lipid level of feed at 8.11, 10.24, 12.28 and 14.33% respectively. Diet without sardine oil (6.05% total lipid) was served as basal treatment. The giant freshwater prawn, Macrobrachium rosenbergii was used as test animal and the experiment was lost for 112 days. Significantly, higher gain in weight of prawn was recorded in the treatment with 6.07% sardine oil incorporation followed by higher specific growth rate, food conversion rate and protein efficiency ratio. The 8.07% sardine oil diet produced the highest RNA: DNA ratio in the prawn muscle. Digestive enzyme analyses in the digestive tract and mid-gut gland showed the greatest activity in prawns fed the 8.07% diet.

Keywords: digestive enzyme, fish diet, Macrobrachium rosenbergii, sardine oil

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Authors: Leixuri Aguirre, Iñaki Milton-Laskibar, Elizabeth Hijona, Luis Bujanda, Agnes M. Rimando, Maria P. Portillo

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Introduction: In recent years great attention has been paid by scientific community to phenolic compounds as active biomolecules naturally present in foodstuffs due to their beneficial effects on health. Pterostilbene is a resveratrol dimethylether derivative which shows higher biodisponibility. Objective. To analyze the effects of two doses of pterostilbene on several markers of thermogenic capacity in a model of genetic obesity, which shows reduced thermogenesis. Methods: The experiment was conducted with thirty Zucker (fa/fa) rats that were distributed in 3 experimental groups, the control group and two groups orally administered with pterostilbene at 15 and 30 mg/kg body weight/day for 6 weeks. Gene expression of Ucp1, Pgc-1α, Cpt1b, Pparα, Nfr1, Tfam and Cox-2 were assessed by RT-PCR, protein expression of UCP1 and GLUT4 by western blot and enzyme activity of carnitine palmitoyl transferase 1b and citrate synthase by spectrophotometry in interscapular brown adipose tissue (iBAT). Statistical analysis was performed by using one way ANOVA and Newman-Keuls as post-hoc test. Results: Pterostilbene did not change gene expression of Pgc-1α. However, significant increases were found in the expression of Ucp1, Pparα, Nfr-1 and Cox-2. Protein expression of UCP1 and GLUT4 was increased in animals treated with pterostilbene, as well as the activities of CPT-1b and CS. These effects were observed with both doses of pterostilbene, without differences between them. Conclusions: These results show that pterostilbene increases thermogenic and oxidative capacity of brown adipose tissue in obese rats. Whether these effects effectively contribute to the anti-obesity properties of these compound needs further research. Acknowledgments: MINECO-FEDER (AGL2015-65719-R), Basque Government (IT-572-13), University of the Basque Country (ELDUNANOTEK UFI11/32), Institut of Health Carlos III (CIBERobn). Iñaki Milton is a fellowship from the Basque Government.

Keywords: brown adipose tissue, pterostilbene, thermogenesis, uncoupling protein 1

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Authors: Wizna, Helmi Muis, Hafil Abbas

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An experiment was conducted to improve the nutrient value of sago pith (Metroxylon sago Rottb) supplemented with Zn, Sulfur and urea through fermentation by using cellulolytic bacteria (Bacillus amyloliquefaciens) as inoculums. The experiment was determination of the optimum dose combination (dosage of Zn, S and urea) for sago pith fermentation based on nutrient quality and quantity of these fermented products. The study was conducted in experimental method, using the completely randomized design in factorial with 3 treatments consist of: factor A (Dose of urea: A1 = 2.0%, A2 = 3.0%), factor B (Dose of S: B1 = 0.2%, B2 = 0.4%) and factor C (Dose of Zn: C1 = 0.0025%, C2 = 0.005%). Results of study showed that optimum condition for fermentation process of sago pith with B. amyloliquefaciens caused a change of nutrient content was obtained at urea (3%), S (0,2%), and Zn (0,0025%). This fermentation process was able to increase amino acid average, reduce crude fiber content by 67% and increase crude protein by 433%, which made the nutritional value of the product based on dry matter was 18.22% crude protein, 12.42% crude fiber, 2525 Kcal/kg metabolic energy and 65.73% nitrogen retention.

Keywords: fermentation, sago pith, sulfur, Zn, urea, Bacillus amyloliquefaciens

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Authors: Apisaraporn Baicharoen, Prapasiri Pongprayoon

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Betaine aldehyde dehydrogenase 2 (BADH2) is an enzyme that inhibits the accumulation of 2-acetyl-1-pyrroline (2AP), a potent flavor compound in rice fragrance. BADH2 contains three domains (NAD-binding, substrate-binding, and oligomerization domains). It catalyzes the oxidation of amino aldehydes. The lack of BADH2 results in the formation of 2AP and consequently an increase in rice fragrance. To date, inadequate data on BADH2 structure and function are available. An insight into the nature of BADH2 can serve as one of key starting points for the production of high quality fragrant rice. In this study, we therefore constructed the homology model of BADH2 and employed 500-ns Molecular Dynamics simulations (MD) to primarily understand the structural and dynamic properties of BADH2. Initially, Ramachandran plot confirms the good quality of modeled protein structure. Principle Component Analysis (PCA) was also calculated to capture the protein dynamics. Among 3 domains, the results show that NAD binding site is found to be more flexible. Moreover, interactions from key amino acids (N162, E260, C294, and Y419) that are crucial for function are investigated.

Keywords: betaine aldehyde dehydrogenase 2, fragrant rice, homology modeling, molecular dynamics simulations

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Authors: Sergio Alejandro Cuevas, Catherine Etchebest, Fernando Luis Barroso Da Silva

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The flavivirus genus has several organisms responsible for generating various diseases in humans. Special in Brazil, Zika (ZIKV), Dengue (DENV) and Yellow Fever (YFV) viruses have raised great health concerns due to the high number of cases affecting the area during the last years. Diagnostic is still a difficult issue since the clinical symptoms are highly similar. The understanding of their common structural/dynamical and biomolecular interactions features and differences might suggest alternative strategies towards differential serological diagnostics and therapeutic intervention. Due to their immunogenicity, the primary focus of this study was on the ZIKV, DENV and YFV non-structural proteins 1 (NS1) protein. By means of computational studies, we calculated the main physical chemical properties of this protein from different strains that are directly responsible for the biomolecular interactions and, therefore, can be related to the differential infectivity of the strains. We also mapped the electrostatic differences at both the sequence and structural levels for the strains from Uganda to Brazil that could suggest possible molecular mechanisms for the increase of the virulence of ZIKV. It is interesting to note that despite the small changes in the protein sequence due to the high sequence identity among the studied strains, the electrostatic properties are strongly impacted by the pH which also impact on their biomolecular interactions with partners and, consequently, the molecular viral biology. African and Asian strains are distinguishable. Exploring the interfaces used by NS1 to self-associate in different oligomeric states, and to interact with membranes and the antibody, we could map the strategy used by the ZIKV during its evolutionary process. This indicates possible molecular mechanisms that can explain the different immunological response. By the comparison with the known antibody structure available for the West Nile virus, we demonstrated that the antibody would have difficulties to neutralize the NS1 from the Brazilian strain. The present study also opens up perspectives to computationally design high specificity antibodies.

Keywords: zika, biomolecular interactions, electrostatic interactions, molecular mechanisms

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Authors: Habib Onsori, Somayeh Akrami, Mohammad Rahmati

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Deafness is the most common sensory disorder with the frequency of 1/1000 in many populations. Mutations in the GJB2 (CX26) gene at the DFNB1 locus on chromosome 13q12 are associated with congenital hearing loss. Approximately 80% of congenital hearing loss cases are recessively inherited and 15% dominantly inherited. Mutations of the GJB2 gene, encoding gap junction protein Connexin 26 (Cx26), are the most common cause of hereditary congenital hearing loss in many countries. This report presents two cases of different mutations from Iranian patients with bilateral hearing loss. DNA studies were performed for the GJB2 gene by PCR and sequencing methods. In one of them, direct sequencing of the gene showed a heterozygous T→C transition at nucleotide 604 resulting in a cysteine to arginine amino acid substitution at codon 202 (C202R) in the fourth extracellular domain (TM4) of the protein. The analyses indicate that the C202R mutation appeared de novo in the proband with a possible dominant effect (GenBank: KF 638275). In the other one, DNA sequencing revealed a compound heterozygous mutation (35delG, 363delC) in the Cx26 gene that is strongly associated with congenital non-syndromic hearing loss (NSHL). So screening the mutations for hearing loss individuals referring to genetics counseling centers before marriage and or pregnancy is recommended.

Keywords: CX26, deafness, GJB2, mutation

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Authors: Nebiyou Masebo

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The severities of land degradation in southern Ethiopia has been increasing due to high population density, replacement of an age-old agroforestry (AF) based agricultural system with monocropping. The consequences of these activities combined with climate change have been impaired soil biota, soil organic carbon (SOC), soil glomalin, soil aggregation and aggregate stability. The AF systems could curb these problems due it is an ecologically and economically sustainable. This study was aimed to determine the effect of agroforestry practices (AFPs) on soil glomalin, soil aggregate stability (SAS), and aggregate association with SOC. Soil samples (from two depth level: 0-30 & 30-60 cm) and woody species were collected from homegarden based agroforestry practice (HAFP), cropland based agroforestry practice (ClAFP), woodlot based agroforestry practice (WlAFP) and trees on soil and water conservation based agroforestry practice (TSWAFP) using systematic sampling. In this study, both easily extractable glomalin related soil protein (EEGRSP) and total glomalin related soil protein (TGRSP) were significantly (p<0.05) higher in HAFP compared to others, with decreasing order HAFP>WlAFP>TSWAFP>ClAFP at upper surface but in subsurface in decreasing order: WlAFP>HAFP>TSWAFP>ClAFP. On the other hand, the macroaggregate fraction of AFPs ranged from 22.64-36.51% where the lowest was in ClAFP, while the highest was in HAFP, moreover, the order for subsurface was also the same but SAS decreased with the increasing of soil depths. The micro-aggregate fraction ranged from 15.9–24.56%, where the lowest was in HAFP, but the highest was in ClAFP. Besides, the association of OC with both macro-and micro-aggregates was greatest in HAFP and followed by WlAFP. The findings also showed that both glomalin and SAS were significantly high with woody species diversity and richness. Thus, AFP with good management practice can play role on maintenance of biodiversity, glomalin content and other soil quality parameters with future implications for a stable ecosystem.

Keywords: agroforestry, soil aggregate stability, glomalin, aggregate-associated carbon, HAFP, ClAFP, WlAFP, TSWAFP.

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Authors: Adedokun Mathew Adewale, Ayandiran Samuel Kola, Adekunle Ibironke

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Pigeon pea (Cajanus cajan) seeds contain about 20–22 percent protein and appreciable amounts of essential amino acids and minerals. Hence, this study evaluated the blood profile of weaner rabbits fed Cajanus cajan meal (CCM) as a replacement for groundnut cake. Forty weaner rabbits of mixed breed aged 5 - 6 weeks were used for the study, which lasted for 8 weeks. The rabbits were randomly allocated to four treatments (10 rabbits per treatment) in a completely randomized design. Four concentrate diets were compounded by direct replacement of groundnut cake with Cajanus cajan meal (CCM) at 0, 50, 75, and 100%, respectively. There were no significant differences (p>0.05) among the mean counts of packed cell volume, red blood cell, haemoglobin, and monocyte. The 75% CCM diet had significantly the highest (p<0.05). However, rabbits fed diets containing CCM had significantly higher (p<0.05) eosinophil than 0%CCM. Rabbits fed diets containing 100%CCM had significantly highest (p<0.05) total protein followed by 0%CCM, 75%CCM, and least 50%CCM, while 0%CCM and 75%CCM diets were significantly higher (p<0.05) in albumin. However, animals fed diets containing CCM had significantly lower (p<0.05) cholesterol content than 0%CCM diet. It could be concluded that Cajanus cajan meal could replace groundnut cake up to 100% in the diets of rabbits without any deleterious effect on the blood profile of the animals.

Keywords: blood profile, groundnut cake, pigeon pea, weaner rabbits

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Authors: Stephen Adeniyi Adefegha, Vitor Mostardeiro, Vera Maria Morsch, Ademir F. Morel, Ivana Beatrice Manica Da Cruz, Sabrina Somacal Maria Rosa Chitolina Schetinger

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Moringa stenopetala is often consumed as food and used in folkloric medicine for the management of several diseases. Purpose: This study was set up in order to assess the effect of aqueous extract of Moringa stenopetala on cell viability and oxidative stress biomarkers in BV-2 microglial cells. Aqueous extracts of M. stenopetala were prepared, lyophilized and reconstituted in 0.5% dimethylsulphoxide (DMSO). Cells were treated with M. stenopetala extracts (0.1 - 100 µg/ml) for cell viability and nitric oxide (NO) production tests. However, M. stenopetala extract (50 µg/ml) was used in the treatment of cells for the determination of protein carbonyl content and reactive oxygen species (ROS) level. Incubation of BV-2 microglia cell with M. stenopetala extract maintained cell viability, diminished NO and ROS levels, and reduced protein carbonyl contents Chlorogenic acid, rutin, kaempferol and quercetin derivatives were the main phenolic compounds identified in M. stenopetala leaf extract. These phenolic compounds present in M. stenopetala may be responsible for the mitigation of oxidative stress in BV-2 microglial cells.

Keywords: oxidative stress, BV-2 microglial cell, Moringa stenopetala, cell viability, antioxidant

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Authors: Tajudin Ahmed

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Malnutrition is a significant problem in developing countries, particularly among children, due to inadequate diets, lack of proper care, and unequal distribution of food within households. High rates of malnutrition have been shown in Ethiopia, including stunting, underweight, and wasting. This study aims to assess the prevalence and associated factors of Protein-Energy Malnutrition (PEM) among children aged 6-59 months in Babile Town. The study utilized a community-based cross-sectional design conducted in Babile Town, Eastern Ethiopia. Two kebeles were randomly selected, and a census was conducted to identify eligible households. A total of 391 households with children aged 6-59 months were included in the study. Data was collected using structured questionnaires, and anthropometric measurements were taken to assess the weight and height of the children. The study found that a majority of the mothers (72.34%) and fathers (43%) had no formal education. Among the mothers who could read and write, a small percentage had completed primary (14%) or secondary (14%) education, and even fewer had higher education (2.7%). Similarly, among the fathers who could read and write, a majority had completed primary (46.15%) or secondary (27.22%) education, with smaller percentages completing preparatory (8.4%) or higher education (6.29%). The prevalence of malnutrition in the study area was high, with 38.85% of children experiencing stunting (8.2% severely stunted), 50.13% wasting (9% severely wasted), and 41.43% underweight (6.65% severely underweight). These findings indicate a significant burden of malnutrition in Babile Town, likely exacerbated by the high prevalence of infectious diseases such as diarrhea. The study concludes that the prevalence of malnutrition, particularly stunting, wasting, and underweight, is high in Babile Town. The findings indicate the urgent need for interventions to address malnutrition and improve nutrition and healthcare practices in the study area. These results can serve as a baseline for future studies and inform policymakers and healthcare providers in their efforts to combat childhood malnutrition.

Keywords: protein-energy malnutrition, children 6-59 month age babble town, Marasmus

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Authors: Yugander Arra, Florence Auguy, Melissa Stiebner, Sophie Chéron, Michael M. Wudick, Van Schepler-Luu, Sébastien Cunnac, Wolf B. Frommer, Laurence Albar

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Rice yellow mottle virus (RYMV) is one of the most important diseases affecting rice in Africa. The most promising strategy to reduce yield losses is the use of highly resistant varieties. The resistance gene RYMV2 is homolog of the Arabidopsis constitutive expression of pathogenesis related protein-5 (AtCPR5) nucleoporin gene. Resistance alleles are originating from African cultivated rice Oryza glaberrima, rarely cultivated, and are characterized by frameshifts or early stop codons, leading to a non-functional or truncated protein. Rice possesses two paralogs of CPR5 and function of these genes are unclear. Here, we evaluated the role of the two rice candidate nucleoporin paralogs OsCPR5.1 (pathogenesis-related gene 5; RYMV2) and OsCPR5.2 by CRISPR/Cas9 genome editing. Despite striking sequence and structural similarity, only loss-of-function of OsCPR5.1 led to full resistance, while loss-of-function oscpr5.2 mutants remained susceptible. Short N-terminal deletions in OsCPR5.1 also did not lead to resistance. In contrast to Atcpr5 mutants, neither OsCPR5.1 nor OsCPR5.2 knock out mutants showed substantial growth defects. Taken together, the candidate nucleoporin OsCPR5.1, but not its close homolog OsCPR5.2, plays a specific role for the susceptibility to RYMV, possibly by impairing the import of viral RNA or protein into the nucleus. Whereas gene introgression from O. glaberrima to high yielding O. sativa varieties is impaired by strong sterility barriers and the negative impact of linkage drag, genome editing of OsCPR5.1, while maintaining OsCPR5.2 activity, thus provides a promising strategy to generate O. sativa elite lines that are resistant to RYMV.

Keywords: CRISPR Cas9, genome editing, knock out mutant, recessive resistance, rice yellow mottle virus

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Authors: Sunidhi Syal, Rasangi Tennakoon, Patrick O'Donoghue

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Polyglutamine (polyQ) diseases are a group of diseases related to neurodegeneration caused by repeats of the amino acid glutamine (Q) in the DNA, which translates into an elongated polyQ tract in the protein. The pathological explanation is that the polyQ tract forms cytotoxic aggregates in the neurons, leading to their degeneration. There are no cures or preventative efforts established for these diseases as of today, although the symptoms of these diseases can be relieved. This study specifically focuses on Huntington's disease, which is a type of polyQ disease in which aggregation is caused by the extended cytosine, adenine, guanine (CUG) codon repeats in the huntingtin (HTT) gene, which encodes for the huntingtin protein. Using this principle, we attempted to create six models, which included mutating wildtype tRNA alanine variant tRNA-AGC-8-1 to have glutamine anticodons CUG and UUG so serine is incorporated at glutamine sites in poly Q tracts. In the process, we were successful in obtaining tAla-8-1 CUG mutant clones in the HTTexon1 plasmids with a polyQ tract of 23Q (non-pathogenic model) and 74Q (disease model). These plasmids were transfected into mouse neuroblastoma cells to characterize protein synthesis and aggregation in normal and mistranslating cells and to investigate the effects of glutamines replaced with alanines on the disease phenotype. Notably, we observed no noteworthy differences in mean fluorescence between the CUG mutants for 23Q or 74Q; however, the Triton X-100 assay revealed a significant reduction in insoluble 74Q aggregates. We were unable to create a tAla-8-1 UUG mutant clone, and determining the difference in the effects of the two glutamine anticodons may enrich our understanding of the disease phenotype. In conclusion, by generating structural disruption with the amino acid alanine, it may be possible to find ways to minimize the toxicity of Huntington's disease caused by these polyQ aggregates. Further research is needed to advance knowledge in this field by identifying the cellular and biochemical impact of specific tRNA variants found naturally in human genomes.

Keywords: Huntington's disease, polyQ, tRNA, anticodon, clone, overlap PCR

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Authors: A. M. S. S. Amarasiri, A. P. Attanayake, K. A. P. W. Jayatilaka, L. K. B. Mudduwa

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Adriamycin (ADR) is an effective anthracyclin antitumor drug, but its clinical use is limited due to renal toxicity. The leaves of Asparagus falcatus (Family: Liliaceae) have been used in the management of renal diseases since antiquity. In the present investigation, the aqueous leaf extract of A. falcatus was evaluated for acute nephroprotective activity in ADR induced nephrotoxic rats. Nephrotoxicity was induced in healthy male Wistar rats by intraperitoneal administration of ADR 20 mg/kg. The lyophilized powder of the aqueous refluxed (4h) leaf extract of A. falcatus was administered orally at three selected doses; 200, 400 and 600 mg/kg for three consecutive days. Fosinopril sodium (0.09 mg/kg) was used as the standard drug. Administration of the plant extract and the standard drug was commenced 24 hours after the induction of nephrotoxicity to rats. The nephroprotective effect was determined by selected biochemical parameters and by the assessment of histopathology on H and E stained kidney sections. The results were compared to a group of control rats with ADR induced nephrotoxicity. A group of rats administered with the equivalent volume of normal saline served as the healthy control. Administration of ADR 20 mg/kg produced a significant increase in the concentrations of serum creatinine (61%) and urine protein (73%) followed by a significant decrease in serum total protein (21%) and albumin (44%) of the plant extract treated animals compared to the healthy control group (p < 0.05). The aqueous extract of Asparagus falcatus at the three doses; 200, 400 and 600 mg/kg and the standard drug were found to decrease the elevation of concentrations of serum creatinine (33%, 51%, 54% and 42%) and urine protein (8%, 63%, 80% and 86%) respectively. The serum concentrations of total protein (12%, 17%, 29% and 12%) and albumin (3%, 17%, 17% and 16%) were significantly increased compared to the nephrotoxic control group respectively. Assessment of histopathology on H and E stained kidney sections demonstrated that ADR induced renal injury, as evidenced by loss of brush border, cytoplasmic vacuolization, pyknosis in renal tubular epithelial cells, haemorrhages, glomerular congestion and presence of hyaline casts. Treatment with the plant extract and the standard drug resulted in attenuation of the morphological destruction in rats. The results of the present study revealed that the aqueous leaf extract of A. falcatus possesses significant nephroprotective activity against adriamycin induced acute nephrotoxicity. The improved kidney functions were supported with the results of selected biochemical parameters and histological changes observed on H and E stained sections of the kidney tissues in Wistar rats.

Keywords: adriamycin induced nephrotoxicity, asparagus falcatus, biochemical assessment, histopathological assessment, nephroprotective activity

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Authors: Agnieszka Sobczak-Kupiec, Dagmara Malina, Klaudia Pluta, Wioletta Florkiewicz, Bozena Tyliszczak

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Introduction: Request for compound of phosphorus grows continuously, thus, it is searched for alternative sources of this element. One of these sources could be by-products from meat industry which contain prominent quantity of phosphorus compounds. Hydroxyapatite, which is natural component of animal and human bones, is leading material applied in bone surgery and also in stomatology. This is material, which is biocompatible, bioactive and osteoinductive. Methodology: Hydroxyapatite preparation: As a raw material was applied deproteinized and defatted bone pulp called bone sludge, which was formed as waste in deproteinization process of bones, in which a protein hydrolysate was the main product. Hydroxyapatite was received in calcining process in chamber kiln with electric heating in air atmosphere in two stages. In the first stage, material was calcining in temperature 600°C within 3 hours. In the next stage unified material was calcining in three different temperatures (750°C, 850°C and 950°C) keeping material in maximum temperature within 3.0 hours. Bone sludge: Bone sludge was formed as waste in deproteinization process of bones, in which a protein hydrolysate was the main product. Pork bones coming from the partition of meat were used as a raw material for the production of the protein hydrolysate. After disintegration, a mixture of bone pulp and water with a small amount of lactic acid was boiled at temperature 130-135°C and under pressure4 bar. After 3-3.5 hours boiled-out bones were separated on a sieve, and the solution of protein-fat hydrolysate got into a decanter, where bone sludge was separated from it. Results of the study: The phase composition was analyzed by roentgenographic method. Hydroxyapatite was the only crystalline phase observed in all the calcining products. XRD investigation was shown that crystallization degree of hydroxyapatite was increased with calcining temperature. Conclusion: The researches were shown that phosphorus content is around 12%, whereas, calcium content amounts to 28% on average. The conducted researches on bone-waste calcining at the temperatures of 750-950°C confirmed that thermal utilization of deproteinized bone-waste was possible. X-ray investigations were confirmed that hydroxyapatite is the main component of calcining products, and also XRD investigation was shown that crystallization degree of hydroxyapatite was increased with calcining temperature. Contents of calcium and phosphorus were distinctly increased with calcining temperature, whereas contents of phosphorus soluble in acids were decreased. It could be connected with higher crystallization degree of material received in higher temperatures and its stable structure. Acknowledgements: “The authors would like to thank the The National Centre for Research and Development (Grant no: LIDER//037/481/L-5/13/NCBR/2014) for providing financial support to this project”.

Keywords: bone by-products, bone sludge, calcination, hydroxyapatite

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Authors: Cheng-Cao Sun, Shu-Jun Li, Cuili Yang, Yongyong Xi, Liang Wang, Feng Zhang, De-Jia Li

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Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4, and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1.

Keywords: hsa-miRNA-326 (miR-326), cyclin D1, non-small cell lung cancer (NSCLC), proliferation, apoptosis

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Authors: Mardiana Ahamad Zabidi, Akmalluddin Md. Yunus

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Soursop (Anona muricata) is one of the underutilized tropical fruits containing nutrients, particularly dietary fibre and antioxidant properties that are beneficial to human health. This objective of this study is to investigate the feasibility of matured soursop pulp flour (SPF) to be substituted with high-protein wheat flour in bread. Bread formulation was substituted with different levels of SPF (0%, 5%, 10% and 15%). The effect on physicochemical properties and sensory attributes were evaluated. Higher substitution level of SPF resulted in significantly higher (p<0.05) fibre, protein and ash content, while fat and carbohydrate content reduced significantly (p<0.05). FESEM showed that the bread crumb surface of control and 5% SPF appeared to distribute evenly and coalesced by thin gluten film. However, higher SPF substitution level in bread formulation exhibited a deleterious effect by formation of discontinuous gluten network. For texture profile analysis, 5% SPF bread resulted in the lowest value of hardness. The score of sensory evaluation showed that 5% SPF bread received good acceptability and is comparable with control bread.

Keywords: soursop pulp flour, bread, physicochemical properties, sensory attributes, scanning electron microscopy (SEM)

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