Search results for: protein engineering

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

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

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

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Authors: Richa Mardianingrum, Srie R. N. Endah

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In this research, we have designed four isoniazid derivatives i.e., isonicotinohydrazide (1-isonicotinoyl semicarbazide, 1-thiosemi isonicotinoyl carbazide, N '-(1,3-dimethyl-1 h-pyrazole-5-carbonyl) isonicotino hydrazide, and N '-(1,2,3- 4-thiadiazole-carbonyl) isonicotinohydrazide. The docking and molecular dynamic have performed to them in order to study its interaction with Mycobacterium tuberculosis Enoyl-Acyl Carrier Protein Reductase (InhA). Based on this research, all of the compounds were predicted to have a stable interaction with Mycobacterium tuberculosis Enoyl-Acyl Carrier Protein Reductase (INHA) receptor, so they could be used as an anti-tuberculosis drug candidate.

Keywords: anti-tuberculosis, docking, Inhibin alpha subunit, InhA, inhibition, synthesis, isonicotinohydrazide

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Authors: Fatima Zohra Bissaad, Farid Bounaceur, Nassima Behidj, Nadjiba Chebouti, Fatma Halouane, Bahia Doumandji-Mitiche

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Effect of Beauveria bassiana and Metarhizium anisopliae var. acridum on the 5^th instar nymphs of Schistocerca gregaria was studied in the laboratory. Infection by these both entomopathogenic fungi caused reduction in the hemolymph total protein. The average amounts of total proteins were 2.3, 2.07, 2.09 µg/100 ml of haemolymph in the control and M. anisopliae var. acridum, and B. bassiana based-treatments, respectively. Three types of haemocytes were recognized and identified as prohaemocytes, plasmatocytes and granulocytes. The treatment caused significant reduction in the total haemocyte count and in each haemocyte type on the 9^th day after its application.

Keywords: Beauveria bassiana, haemolymph picture, haemolymph protein, Metarhizium anisopliae var. acridum, Schistocerca gregaria

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Authors: Lee A. Browne, K. Rumbold

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The first fluorinating enzyme, 5'-fluoro-5'-deoxyadenosine synthase (fluorinase) was isolated from the soil bacterium Streptomyces cattleya. Such an enzyme, with the ability to catalyze a C-F bond, presents great potential as a biocatalyst. Naturally fluorinated compounds are extremely rare in nature. As a result, the number of fluorinases identified remains relatively few. The field of fluorination is almost completely synthetic. However, with the increasing demand for fluorinated organic compounds of commercial value in the agrochemical, pharmaceutical and materials industries, it has become necessary to utilize biologically based methods such as biocatalysts. A key step in this crucial process is the large-scale production of the fluorinase enzyme in considerable quantities for industrial applications. Thus, this study aimed to optimize expression of the fluorinase enzyme in both prokaryotic and eukaryotic expression systems in order to obtain high protein yields. The fluorinase gene was cloned into the pET 41b(+) and pPinkα-HC vectors and used to transform the expression hosts, E.coli BL21(DE3) and Pichia pastoris (PichiaPink™ strains) respectively. Expression trials were conducted to select optimal conditions for expression in both expression systems. Fluorinase catalyses a reaction between S-adenosyl-L-Methionine (SAM) and fluoride ion to produce 5'-fluorodeoxyadenosine (5'FDA) and L-Methionine. The activity of the enzyme was determined using HPLC by measuring the product of the reaction 5'FDA. A gradient mobile phase of 95:5 v/v 50mM potassium phosphate buffer to a final mobile phase containing 80:20 v/v 50mM potassium phosphate buffer and acetonitrile were used. This resulted in the complete separation of SAM and 5’-FDA which eluted at 1.3 minutes and 3.4 minutes respectively. This proved that the fluorinase enzyme was active. Optimising expression of the fluorinase enzyme was successful in both E.coli and PichiaPink™ where high expression levels in both expression systems were achieved. Protein production will be scaled up in PichiaPink™ using fermentation to achieve large-scale protein production. High level expression of protein is essential in biocatalysis for the availability of enzymes for industrial applications.

Keywords: biocatalyst, expression, fluorinase, PichiaPink™

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Authors: Soo Dong Cho, In-Ohk Ouh, Byeong Sul Kang, Seyeon Park, In-Soo Cho, Jae Young Song

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An VP2 gene from the current prevalent CPV (Canine Parvovirus) strain (new CPV-2a) in the Republic of Korea was expressed in a baculovirus expression system. Genomic DNA was extracted from the isolate strain CPV-2a. The recombinant baculovirus, containing the coding sequences of VP2 with the histidine tag at the N-terminus, were generated by using the Bac-to-Bac system. For production of the recombinant VP2 proteins, SF9 cells were transfection into 6 wells. Propagation of recombinant baculoviruses and expression of the VP2 protein were performed in the Sf9 cell line maintained. The proteins were detected to Western blot anlaysis. CPV-2a VP2 was detected by Western blotting the monoclonal antibodies recognized 6x His and the band had a molecular weight of 65 KDa. We demonstrated that recombinant CPV-2a VP2 expression in baculovirus. The recombinant CPV-2a VP2 may able to development of specific diagnostic test and vaccination of against CPV2. This study provides a foundation for application of CPV2 on the development of new CPV2 subunit vaccine.

Keywords: baculovirus, canine parvovirus 2a, Dog, Korea

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Authors: A. M. Nour, M. A. Zaki, E. A. Omer, Nourhan Mohamed

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Performances of mixed-sex Nile tilapia (Oreochromis niloticus) fingerlings (11.33 ± 1.78 g /fish) reared under biofloc system developed by molasses and sucrose as carbon sources in indoor fiberglass tanks were evaluated. Six indoor fiberglass tanks (1m 3 each filled with 1000 l of underground fresh water), each was stocked with 2kg fish were used for 14 weeks experimental period. Three experimental groups were designed (each group 2 tanks) as following: 1-control: 20% daily without biofloc, 2-zero water exchange rate with biofloc (molasses as C source) and 3-zero water exchange rate with biofloc (sucrose as C source). Fish in all aquariums were fed on floating feed pellets (30% crude protein, 3 mm in diameter) at a rate of 3% of the actual live fish body, 3 times daily and 6 days a week. Carbohydrate supplementations were applied daily to each tank two hrs, after feeding to maintain the carbon: nitrogen ratio (C: N) ratio 20:1. Fish were reared under continuous aeration by pumping air into the water in the tank bottom using two sandy diffusers and constant temperature between 27.0-28.0 ºC by using electrical heaters for 10 weeks. Criteria's for assessment of water quality parameters, biofloc production and fish growth performances were collected and evaluated. The results showed that total ammonia nitrogen in control group was higher than biofloc groups. The biofloc volumes were 19.13 mg/l and 13.96 mg/l for sucrose and molasses, respectively. Biofloc protein (%), ether extract (%) and gross energy (kcal/100g DM), they were higher in biofloc molasses group than biofloc sucrose group. Tilapia growth performances were significantly higher (P < 0.05) with molasses group than in sucrose and control groups, respectively. The highest feed and nutrient utilization values for protein efficiency ratio (PER), protein productive (PPV%) and energy utilization (EU, %) were higher in molasses group followed by sucrose group and control group respectively.

Keywords: biofloc, Nile tilapia, cabohydrates, performances

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Authors: Patcharakamon Nooeaid, Piyachat Chuysrinuan

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Electrospun natural polymers-based nanofibers are one of the most interesting materials used in tissue engineering and drug delivery applications. Bead-on-string nanofibers have gained considerable interest for sustained drug release. Vancomycin was used as the model drug and sodium alginate (SA)/soy protein isolated (SPI) as the polymer blend to fabricate the bead-on-string nanofibers by aqueous-based electrospinning. The bead-on-string SA/SPI nanofibers were successfully fabricated by the addition of poly(ethylene oxide) (PEO) as a co-blending polymer. SA-PEO with mass ratio of 70/30 showed the best spinnability with continuous nanofibers without the occurrence of beads. Bead structure formed with the addition of SPI and bead number increased with increasing SPI content. The electrospinning of 80/20 SA-PEO/SPI was obtained as a great promising bead-on-string nanofibers for drug loading, while the solution of 50/50 was not able to obtain continuous fibers. In vitro release tests showed that a more sustainable release profile up to 14 days with less initial burst release on day 1 could be obtained from the bead-on-string fibers than from smooth fibers with uniform diameter. In addition, vancomycin-loaded beaded fibers inhibited the growth of Staphylococcus aureus (S. aureus) bacteria. Therefore, the SA-PEO/SPI nanofibers showed the potential to be used as biomaterials for tissue engineering and drug delivery.

Keywords: bead-on-string fibers, electrospinning, drug delivery, tissue engineering

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Authors: Nafees Ahmed, Nur Izyani Kamaruzman, Saralla Nathan, Mohd Ezharul Hoque Chowdhury, Anuar Zaini Md Zain, Iekhsan Othman, Sharifah Binti Syed Hassan

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Meat quality is always subject to consumer scrutiny when purchasing from retail markets on mislabeling as fresh meat. Various physiological and biochemical changes influence the quality of meat. As a major component of muscle tissue, proteins play a major role in muscle foods. In meat industry, freezing is the most common form of storage of meat products. Repeated cycles of freezing and thawing are common in restaurants, kitchen, and retail outlets and can also occur during transportation or storage. Temperature fluctuation is responsible for physical, chemical, and biochemical changes. Repeated cycles of ‘freeze-thaw’ degrade the quality of meat by stimulating the lipid oxidation and surface discoloration. The shelf life of meat is usually determined by its appearance, texture, color, flavor, microbial activity, and nutritive value and is influenced by frozen storage and subsequent thawing. The main deterioration of frozen meat during storage is due to protein. Due to the large price differences between fresh and frozen–thawed meat, it is of great interest to consumer to know whether a meat product is truly fresh or not. Researchers have mainly focused on the reduction of moisture loss due to freezing and thawing cycles of meat. The water holding capacity (WHC) of muscle proteins and reduced water content are key quality parameters of meat that ultimately changes color and texture. However, there has been limited progress towards understanding the actual mechanisms behind the meat quality changes under the freeze–thaw cycles. Furthermore, effect of freeze-thaw process on integrity of proteins is ignored. In this paper, we have studied the effect of ‘freeze-thawing’ on physicochemical changes of chicken meat protein. We have assessed the quality of meat by pH, spectroscopic measurements, Western Blot. Our results showed that increase in freeze-thaw cycles causes changes in pH. Measurements of absorbance (UV-visible and IR) indicated the degradation of proteins. The expression of various proteins (CREB, AKT, MAPK, GAPDH, and phosphorylated forms) were performed using Western Blot. These results indicated the repeated cycles of freeze-thaw is responsible for deterioration of protein, thus causing decrease in nutritious value of meat. It damges the use of these products in Islamic Sharia.

Keywords: chicken meat, freeze-thaw, halal, protein, western blot

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Authors: Lucia Salvioni, Pietro Giorgio Lovaglio, Valerio Leoni, Miriam Colombo, Luisa Fiandra

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The involvement of plasmatic surfactant protein-D (SP-D) in pulmonary diseases has been long investigated, and over the last two years, more interest has been directed to determine its role as a marker of COVID-19. In this direction, several studies aimed to correlate pulmonary surfactant proteins with the clinical manifestations of the virus indicated SP-D as a prognostic biomarker of COVID-19 pneumonia severity. The present work has performed a retrospective study on a relatively large cohort of patients of Hospital Pio XI of Desio (Lombardia, Italy) with the aim to assess differences in the hematic SP-D concentrations among COVID-19 patients and healthy donors and the role of SP-D as a prognostic marker of severity and/or of mortality risk. The obtained results showed a significant difference in the mean of log SP-D levels between COVID-19 patients and healthy donors, so as between dead and survived patients. SP-D values were significantly higher for both hospitalized COVID-19 and dead patients, with threshold values of 150 and 250 ng/mL, respectively. SP-D levels at admission and increasing differences among follow-up and admission values resulted in the strongest significant risk factors of mortality. Therefore, this study demonstrated the role of SP-D as a predictive marker of SARS-CoV-2 infection and its outcome. A significant correlation of SP-D with patient mortality indicated that it is also a prognostic factor in terms of mortality, and its early detection should be considered to design adequate preventive treatments for COVID-19 patients.

Keywords: SARS-CoV-2 infection, COVID-19, surfactant protein-D (SP-D), mortality, biomarker

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Authors: Hajar Zarei, AliAkbar Zarenejadatashgah, Vuk Uskoković, Hiroshi Watabe

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The reactive oxygen species (ROS) generated by radiation in nuclear diagnostic imaging and radiotherapy could damage the structure of the proteins in noncancerous cells surrounding the tumor. The critical factor in many age-related diseases, such as Alzheimer, Parkinson, or Huntington diseases, is the oxidation of proteins by the ROS as molecular triggers of the given pathologies. Our studies by spectroscopic experiments showed doses close to therapeutic ones (1 to 5 Gy) could lead to changes of secondary and tertiary structures in BSA protein macromolecule as a protein model as well as the aggregation of polypeptide chain but without the fragmentation. For this reason, we investigated the radioprotective effects of natural (vitamin C and E) and synthetic materials (CNPs and FIOMPs) on the structural changes in BSA protein induced by gamma irradiation at a therapeutic dose (3Gy). In the presence of both vitamins and synthetic materials, the spectroscopic studies revealed that irradiated BSA was protected from the structural changes caused by ROS, according to in vitro research. The radioprotective property of CNPs and FIOMPs arises from enzyme mimetic activities (catalase, superoxide dismutase, and peroxidase) and their antioxidant capability against hydroxyl radicals. In the case of FIOMPs, a porous structure also leads to increased ROS recombination with each other in the same radiolytic track and subsequently decreased encounters with BSA. The hydrophilicity of vitamin C resulted in the major scavenging of ROS in the solvent, whereas hydrophobic vitamin E localized on the nonpolar patches of the BSA surface, where it did not only neutralize them thanks to the moderate BSA binding constant but also formed a barrier for diffusing ROS. To the best of our knowledge, there has been a persistent lack of studies investigating the radioactive effect of mentioned materials on proteins. Therefore, the results of our studies can open a new widow for application of these common dietary ingredients and new synthetic NPs in improving the safety of radiotherapy.

Keywords: reactive oxygen species, spectroscopy, bovine serum albumin, gamma radiation, radioprotection

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Authors: S. Yezli-Touiker, L. Kirane-Amrani, N. Soltani-Mazouni

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Ephestia kuehniella Zeller Lepidoptera, Pyralidae commonly called Mediterranean flour moth, is serious cosmopolitan pest of stored grain products, particularly flour Month. This species is also a source of allergen that causes asthma and rhinitis. Captopril is an inhibitor of angiotensin converting enzyme (ACE) it was tested in vivo by topical application on development of E. kuehniella. The compound is diluted in acetone and applied topically to newly emerged pupae (10mg/2ml). Report chitin protein of cuticule and ecdysteroid Amounts were determined in vivo. Results show that the captopril does not affect chitin protein of cuticule but traitment with captopril increase the hormonal production, the quantitative analysis reveals the presence of two peaks one at third and another at fifth day.

Keywords: Ephestia kuehniella, cuticule, hormone, captopril

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Authors: Onur Gönülal, Zafer Ceylan, Gülgün F. Unal Sengor

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The differences of Parapenaus longirostris caught from The North Aegean Sea and the Marmara Sea on proximate composition, sensorial analysis (for raw and cooked samples), flesh productivity of the samples were investigated. The moisture, protein, lipid, ash, carbohydrate, energy contents of shrimp caught from The North Aegean Sea were 74.92 ± 0.1, 20.32 ± 0.16, 2.55 ± 0.1, 2.13 ± 0.08, 0.08, 110.1 kcal/100g, respectively. The moisture, protein, lipid, ash, carbohydrate, energy contents of shrimp caught from Marmara Sea were 76.9 ± 0.02, 19.06 ± 0.03, 2.22 ± 0.08, 1.51 ± 0.04, 0.33, 102.77 kcal/100g, respectively. The protein, lipid, ash and energy values of the Northern Aegean Sea shrimp were higher than The Marmara Sea shrimp. On the other hand, The moisture, carbohydrate values of the Northern Aegean Sea shrimp were lower than the other one. Sensorial analysis was done for raw and cooked samples. Among all properties for raw samples, flesh color, shrimp connective tissue, shrimp body parameters were found different each other according to the result of the panel. According to the result of the cooked shrimp samples among all properties, cooked odour, flavours, texture were found to be different from each other, as well. Especially, flavours and textural properties of cooked shrimps of the Northern Aegean Sea were higher than the Marmara Sea shrimp. Flesh productivity of Northern Aegean Sea shrimp was found as 46.42 %, while that of the Marmara Sea shrimp was found as 47.74 %.

Keywords: shrimp, biological differences, proximate value, sensory, Parapenaus longirostris, flesh productivity

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Authors: Seyed Mohammad Ali Zahedi

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This field experiment was conducted in Isfahan in 2011 in order to study the effect of plant density and planting pattern on growth, yield and quality of silage corn (SC 704) using a randomized complete block design with split plot layout and four replications. The main plot consisted of three planting patterns (60 and 75 cm single planting row and 75 cm double planting row referred to as 60S, 75S and 75T, respectively). The subplots consisted of four levels of plant densities (65000, 80000, 95000 and 110000 plants per hectare). Each subplot consisted of 7 rows, each with 10m length. Vegetative and reproductive characteristics of plants at silking and hard dough stages (when the plants were harvested for silage) were evaluated. Results of variance analysis showed that the effects of planting pattern and plant density were significant on leaf area per plant, leaf area index (at silking), plant height, stem diameter, dry weights of leaf, stem and ear in silking and harvest stages and on fresh and dry yield, dry matter percentage and crude protein percentage at harvest. There was no planting pattern × plant density interaction for these parameters. As row space increased from 60 cm with single planting to 75 cm with single planting, leaf area index and plant height increased, but leaf area per plant, stem diameter, dry weight of leaf, stem and ear, dry matter percentage, dry matter yield and crude protein percentage decreased. Dry matter yield reduced from 24.9 to 18.5 t/ha and crude protein percentage decreased from 6.11 to 5.60 percent. When the plant density increased from 65000 to 110000 plant per hectare, leaf area index, plant height, dry weight of leaf, stem and ear and dry matter yield increased from 19.2 to 23.3 t/ha, whereas leaf area per plant, stem diameter, dry matter percentage and crude protein percentage decreased from 6.30 to 5.25. The best results were obtained with 60 cm row distance with single planting and 110000 plants per hectare.

Keywords: silage corn, plant density, planting pattern, yield

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Authors: Jone Ibarruri, Mikel Manso, Marta Cebrián

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A high amount of food is lost or discarded in the World every year. In addition, in the last decades, an increasing demand of new alternative and sustainable sources of proteins and other valuable compounds is being observed in the food and feeding sectors and, therefore, the use of food by-products as nutrients for these purposes sounds very interesting from the environmental and economical point of view. However, the direct use of discarded fruit and vegetables that present, in general, a low protein content is not interesting as feeding ingredient except if they are used as a source of fiber for ruminants. Especially in the case of aquaculture, several alternatives to the use of fish meal and other vegetable protein sources have been extensively explored due to the scarcity of fish stocks and the unsustainability of fishing for these purposes. Fish mortality is also of great concern in this sector as this problem highly reduces their economic feasibility. So, the development of new functional and natural ingredients that could reduce the need for vaccination is also of great interest. In this work, several fermentation tests were developed at lab scale using a selected mixture of fruit and vegetable discards from a wholesale market located in the Basque Country to increase their protein content and also to produce some bioactive extracts that could be used as additives in aquaculture. Fruit and vegetable mixtures (60/40 ww) were centrifugated for humidity reduction and crushed to 2-5 mm particle size. Samples were inoculated with a selected Rhizopus oryzae strain and fermented for 7 days in controlled conditions (humidity between 65 and 75% and 28ºC) in Petri plates (120 mm) by triplicate. Obtained results indicated that the final fermented product presented a twofold protein content (from 13 to 28% d.w). Fermented product was further processed to determine their possible functionality as a feed additive. Extraction tests were carried out to obtain an ethanolic extract (60:40 ethanol: water, v.v) and remaining biomass that also could present applications in food or feed sectors. The extract presented a polyphenol content of about 27 mg GAE/gr d.w with antioxidant activity of 8.4 mg TEAC/g d.w. Remining biomass is mainly composed of fiber (51%), protein (24%) and fat (10%). Extracts also presented antibacterial activity according to the results obtained in Agar Diffusion and to the Minimum Inhibitory Concentration (MIC) tests determined against several food and fish pathogen strains. In vitro, digestibility was also assessed to obtain preliminary information about the expected effect of extraction procedure on fermented product digestibility. First results indicated that remaining biomass after extraction doesn´t seem to improve digestibility in comparison to the initial fermented product. These preliminary results show that fermented fruit and vegetables can be a useful source of functional ingredients for aquaculture applications and a substitute of other protein sources in the feeding sector. Further validation will be also carried out through “in vivo” tests with trout and bass.

Keywords: fungal solid state fermentation, protein increase, functional extracts, feed ingredients

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Authors: Samson A. Oyeyinka, Taiwo Tijani, Adewumi T. Oyeyinka, Mutiat A. Balogun, Fausat L. Kolawole, John K. Joseph

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Bambara groundnut is an underutilised leguminous crop that has a similar composition to cowpea. Hence, it could be used in making traditional snack usually produced from cowpea paste. In this study, akara egbe, a traditional snack was prepared from Bambara groundnut flour or paste. Cowpea was included as the reference sample. The proximate composition and functional properties of the flours were studies as well as the proximate composition and sensory properties of the resulting akara egbe. Protein and carbohydrate were the main components of Bambara groundnut and cowpea grains. Ash, fat and fiber contents were low. Bambara groundnut flour had higher protein content (23.71%) than cowpea (19.47%). In terms of functional properties, the oil absorption capacity (0.75 g oil/g flour) of Bambara groundnut flour was significantly (p ≤ 0.05) lower than that of the cowpea (0.92 g oil/g flour), whereas, Cowpea flour absorbed more water (1.59 g water/g flour) than Bambara groundnut flour (1.12 g/g). The packed bulk density (0.92 g/mL) of Bambara groundnut was significantly (p ≤ 0.05) higher than cowpea flour (0.82 g/mL). Akara egbe prepared from Bambara groundnut flour showed significantly (p ≤ 0.05) higher protein content (23.41%) than the sample made from Bambara groundnut paste (19.35%). Akara egbe prepared from cowpea paste had higher ratings in aroma, colour, taste, crunchiness and overall acceptability than those made from cowpea flour or Bambara groundnut paste or flour. Bambara groundnut can produce akara egbe with comparable nutritional and sensory properties to that made from cowpea.

Keywords: Bambara groundnut, Cowpea, Snack, Sensory properties

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Authors: Mario Pérez-Won, Vilbett Briones L., Guido Trautmann, María José Bugueño, Gipsy Tabilo-Munizaga, Luis Gonzalez-Cavieres

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The fishing industry generates a significant amount of shrimp byproducts, which often result in environmental contamination. Protein extraction from these by-products is a potential solution to minimize waste and revalue the by-products. To improve the extraction of proteins (by chemical method) from shrimp (Pleuroncodes monodon) by-products, the emerging technologies of ohmic heating (OH), microwaves (MW) and pulsed electric fields (PEF) were used. The results show that microwaves, electrical pulses, and ohmic heating improved performance by 28.19%, 19.25%, and 3.65%, respectively. Furthermore, conformational changes were studied by DSC and FTIR. Subsequently, the use of these proteins in electrospinning technology was evaluated. In conclusion, this study demonstrates that the application of emerging technologies, can significantly improve the extraction yield of proteins from shrimp by-products.

Keywords: electrospinning, emerging technologies, improving extraction, shrimp by-products

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Authors: Kinga Mylkie, Pawel Nowak, Marta Z. Borowska

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The most important proteins in human serum responsible for drug binding are human serum albumin (HSA) and α1-acid glycoprotein (AGP). The AGP molecule is a glycoconjugate containing a single polypeptide chain composed of 183 amino acids (the core of the protein), and five glycan branched chains (sugar part) covalently linked by an N-glycosidic bond with aspartyl residues (Asp(N) -15, -38, -54, -75, - 85) of polypeptide chain. This protein plays an important role in binding alkaline drugs, a large group of drugs used in psychiatry, some acid drugs (e.g., coumarin anticoagulants), and neutral drugs (steroid hormones). The main goal of the research was to obtain magnetic nanoparticles coated with biopolymers in a chemically modified form, which will have highly reactive functional groups able to effectively immobilize the glycoprotein (acid α1-glycoprotein) without losing the ability to bind active substances. The first phase of the project involved the chemical modification of biopolymer starch. Modification of starch was carried out by methods of organic synthesis, leading to the preparation of a polymer enriched on its surface with aldehyde groups, which in the next step was coupled with 3-aminophenylboronic acid. Magnetite nanoparticles coated with starch were prepared by in situ co-precipitation and then oxidized with a 1 M sodium periodate solution to form a dialdehyde starch coating. Afterward, the reaction between the magnetite nanoparticles coated with dialdehyde starch and 3-aminophenylboronic acid was carried out. The obtained materials consist of a magnetite core surrounded by a layer of modified polymer, which contains on its surface dihydroxyboryl groups of boronic acids which are capable of binding glycoproteins. Magnetic nanoparticles obtained as carriers for plasma protein immobilization were fully characterized by ATR-FTIR, TEM, SEM, and DLS. The glycoprotein was immobilized on the obtained nanoparticles. The amount of mobilized protein was determined by the Bradford method.

Keywords: glycoproteins, immobilization, magnetic nanoparticles, polysaccharides

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Authors: Ankush Verma, Rohit Bishist

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The present investigation was carried out at different locations of Shimla and Kinnaur district and nutrient analysis was done in the laboratory of Department of Silviculture and Agroforestry, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Distt. Solan, Himachal Pradesh during 2019-2020 with the objectives to study the seasonal variation in the nutritive value of different Quercus species and to study the farmers’ preference rating of fodder tress species. From each location leaf samples were collected at 3 months interval from each Quercus spp. The findings of the present study revealed that the nutritional traits of leaves of different Quercus species varied among different seasons throughout the year. The dry matter (61.12 to 64.99%), ether extract (4.07 to 4.42%), crude fibre (34.38 to 37.85%), neutral detergent fibre (57.70 to 61.54%), acid detergent fibre (44.64 to 48.51%), total ash (3.57 to 3.91%), acid insoluble ash (44.64 to 48.51%) and calcium (1.31 to 1.53%) increased with the maturity in the leaves of different Quercus species. While, crude protein (9.10 to 10.61%), nitrogen free extract (44.73 to 47.41%), organic matter (96.09 to 96.43%), and phosphorus (0.16 to 0.31%) decreased with the advancing maturity in the leaves of different Quercus species. Maximum mean values for dry matter (65.05%), ether extract (4.45%), crude fibre (40.82%), neutral detergent fibre (61.48%), acid detergent fibre (48.44%), and organic matter (96.67%) among different Quercus species were recorded in Quercus ilex, while, Maximum mean values for crude protein (10.54%), nitrogen free extract (50.53%), total ash (4.05%), acid insoluble ash (0.59%), calcium (1.61%) and phosphorus (0.40%) were recorded in Quercus leucotrichophora.

Keywords: nutritional evaluation, fodder species, crude protein, carbohydrates

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Authors: Don Buddika Oshadi Malaweera, Lora Harris, Bruce Rathgeber, Chibuike C. Udenigwe, Kirsti Rouvinen-Watt

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Fatty liver disease is among the three most severe health concerns for mink and believed to occur through the same mechanism as nursing sickness. In North America, nursing sickness affects about 45% of mink farms and in Canada, approximately 50,000 mink females is affected annually. Orotic acid (OA) plays a critical role in lipid metabolism and can increase hepatic lipids by enhancing Sterol regulatory element binding protein-1c expression and decreasing Carnitine palmitoyl transferase I activity. This study was conducted to identify particular pathways and regulatory control points involved in fatty liver development, and evaluate the effectiveness of arginine and bioactive peptides for prevention and treatment of fatty liver disease in mink. A total of 45 mink were used in 9 treatments. The experimental diets consisted of 1% OA, 2% L-arginine and 5% of whey protein hydrolysates. At the end of 10 days of experimental period, the mink were anaesthetized, sampled for blood and euthanized, samples were obtained for histological, biochemical and molecular assays. The blood samples will be analyzed for clinical chemistry and triacylglycerol. The liver samples will be analyzed for total lipid content and analyzed for 6 genes of interest involved in adipogenic transformation, ER stress, and liver inflammation.

Keywords: fatty liver, L-arginine, mink, orotic acid, whey protein hydrolysates

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Authors: Mahdi Nasiri Tabrizi, A. Dadkhah, M. Khirkhah

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In order to study the effect of planting on yield, yield components and quality traits in bread and durum wheat varieties, a field split-plot experiment based on complete randomized design with three replications was conducted in Agricultural and Natural Resources Research Center of Razavi Khorasan located in city of Mashhad during 2013-2014. Main factor were consisted of five sowing dates (first October, fifteenth December, first March, tenth March, twentieth March) and as sub-factors consisted of different bread wheat (Bahar, Pishgam, Pishtaz, Mihan, Falat and Karim) and two durum wheat (Dena and Dehdasht). According to results of analysis variance the effect of planting date was significant on all examined traits (grain yield, biological yield, harvest index, number of grain per spike, thousands kernel weight, number of spike per square meter, plant height, the number of days to heading, the number of days to maturity, during the grain filling period, percentage of wet gluten, percentage of dry gluten, gluten index, percentage of protein). By delay in planting, majority of traits significantly decreased, except quality traits (percentage of wet gluten, percentage of dry gluten and percentage of protein). Results of means comparison showed, among planting date the highest grain yield and biological yield were related to first planting date (Octobr) with mean of production of 5/6 and 1/17 tons per hectare respectively and the highest bread quality (gluten index) with mean of 85 and percentage of protein with mean of 13% to fifth planting date also the effect of genotype was significant on all traits. The highest grain yield among of studied wheat genotypes was related to Dehdasht cultivar with an average production of 4.4 tons per hectare. The highest protein percentage and bread quality (gluten index) were related to Dehdasht cultivar with 13.4% and Falat cultivar with number of 90 respectively. The interaction between cultivar and planting date was significant on all traits and different varieties had different trend for these traits. The highest grain yield was related to first planting date (October) and Falat cultivar with an average of production of 6/7 tons per hectare while in grain yield did not show a significant different with Pishtas and Mihan cultivars also the most of gluten index (bread quality index) and protein percentage was belonged to the third planting date and Karim cultivar with 7.98 and Dena cultivar with 7.14% respectively.

Keywords: yield component, yield, planting date, cultivar, quality traits, wheat

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Authors: Fitri Rahmi Fadhilah, Edhyana Sahiratmadja, Ani Melani Maskoen, Ratu Safitri, Supartini Syarif, Herman Susanto

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Cervical cancer is the second most common cancer in women after breast cancer. In Indonesia, the incidence of cervical cancer cases is estimated at 25-40 per 100,000 women per year. Human papillomavirus (HPV) infection is a major cause of cervical cancer, and HPV-16 is the most common genotype that infects the cervical tissue. The major late protein L1 may be associated with infectivity and pathogenicity and its variation can be used to classify HPV isolates. The aim of this study was to determine the phylogenetic tree of HPV 16 L1 gene from cervical cancer patient isolates in Bandung. After confirming HPV-16 by Linear Array Genotyping Test, L1 gene was amplified using specific primers and subject for sequencing. Phylogenetic analysis revealed that HPV 16 from Bandung was in the subgroup of Asia and East Asia, showing the close host-agent relationship among the Asian type.

Keywords: L1 HPV 16, cervical cancer, bandung, phylogenetic

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Authors: Sajjad Rezaei, Mahdieh Molanouri Shamsi, Azadeh Jamali

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

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

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Authors: Anjani Kumar Tiwari, Neelam Kumari, Anil Mishra

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The 18-kDa translocator protein (TSPO) previously called as peripheral benzodiazepine receptor, is proven biomarker for variety of neuroinflammation. TSPO is tryptophane rich five transmembranal protein found on outer mitochondrial membrane of steroid synthesising and immunomodulatory cells. In case of neuronal damage or inflammation the expression level of TSPO get upregulated as an immunomodulatory response. By utilizing Benzoxazolone as a basic scaffold, series of TSPO ligands have been designed followed by their screening through in silico studies. Synthesis has been planned by employing convergent methodology in six high yielding steps. For the synthesized ligands the ‘in vitro’ assay was performed to determine the binding affinity in term of Ki. On ischemic rat brain, autoradiography studies were also carried to check the specificity and affinity of the designed radiolabelled ligand for TSPO.Screening was performed on the basis of GScore of CADD based schrodinger software. All the modified and better prospective compound were successfully carried out and characterized by spectroscopic techniques (FTIR, NMR and HRMS). In vitro binding assay showed best binding affinity Ki = 6.1+ 0.3 for TSPO over central benzodiazepine receptor (CBR) Ki > 200. ARG studies indicated higher uptake of two analogues on the lesion side compared with that on the non-lesion side of ischemic rat brains. Displacement experiments with unlabelled ligand had minimized the difference in uptake between the two sides which indicates the specificity of the ligand towards TSPO receptor.

Keywords: TSPO, PET, imaging, Acetamidobenzoxazolone

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Authors: Serap Pektas

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Ataxia telangiectasia mutated (ATM) is a serine-threonine kinase, which is the major regulator of the DNA damage response. ATM is activated upon the formation of DNA double-strand breaks (DSBs) in the cells. ATM phosphorylates the proteins involved in apoptotic responses, cell cycle checkpoint control, DNA repair, etc. Tumor protein p53, known as p53 is one of these proteins that phosphorylated by ATM. Phosphorylation of p53 at Ser15 residue leads to p53 stabilization in the cells. Often enzymes activity is affected by hydrogen ion concentration (pH). In order to find the optimal pH range for ATM activity, steady-state kinetic assays were performed at acidic and basic pH ranges. Ser15 phosphorylation of p53 is determined by using ELISA. The results indicated that the phosphorylation rate was better at basic pH range compared with the acidic pH range. This could be due to enzyme stability, or enzyme-substrate interaction is pH dependent.

Keywords: ataxia telangiectasia mutated, DNA double strand breaks, DNA repair, tumor protein p53

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Authors: Anita Singh, Richa Naula, Manoj Raghav

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Nutrient rich and high-yielding varieties of fenugreek can be developed by using genotypes which are naturally high in nutrients. Gene banks harbour scanty germplasm collection of Trigonella spp. and a very little background information about its genetic diversity. The extent of genetic diversity in a specific breeding population depends upon the genotype included in it. The present investigation aims at the estimation of macronutrient (phosphorus by spectrophotometer and potassium by flame photometer), micronutrients, namely, iron, zinc, manganese, and copper from seeds of fenugreek genotypes using atomic absorption spectrophotometer, protein by Rapid N Cube Analyser and Steroidal Saponins. Twenty-eight genotypes of fenugreek along with two standard checks, namely, Pant Ragini and Pusa Early Bunching were collected from different parts of India, and nutrient contents of each genotype were determined at G. B. P. U. A. & T. Laboratory, Pantnagar. Highest potassium content was observed in PFG-35 (1207 mg/100g). PFG-37 and PFG-20 were richest in phosphorus, iron and manganese content among all the genotypes. The lowest zinc content was found in PFG-26 (1.19 mg/100g), while the maximum zinc content was found in PFG- 28 (4.43 mg/100g). The highest content of copper was found in PFG-26 (1.97 mg/100g). PFG-39 has the highest protein content (29.60 %). Significant differences were observed in the steroidal saponin among the genotypes. Saponin content ranged from 0.38 g/100g to 1.31 g/100g. Steroidal Saponins content was found the maximum in PFG-36 (1.31 g/100g) followed by PFG-17 (1.28 g/100g). Therefore, the genotypes which are rich in nutrient and oil content can be used for plant biofortification, dietary supplements, and herbal products.

Keywords: genotypes, macronutrients, micronutrient, protein, seeds

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Authors: Benito Minjarez, Jesse Haramati, Yury Rodriguez-Yanez, Florencio Recendiz-Hurtado, Juan-Pedro Luna-Arias, Salvador Mena-Munguia

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During the last decades, the world has experienced a rapid industrialization and an expanding economy favoring a demographic boom. As a consequence, countries around the world have focused on developing new strategies related to the production of different farm products in order to meet future demands. Consequently, different strategies have been developed seeking to improve the major food products for both humans and livestock. Corn, after wheat and rice, is the third most important crop globally and is the primary food source for both humans and livestock in many regions around the globe. In addition, maize (Zea mays) is an important source of protein accounting for up to 60% of the daily human protein supply. Generally, many of the cereal grains have proteins with relatively low nutritional value, when they are compared with proteins from meat. In the case of corn, much of the protein is found in the endosperm (75 to 85%) and is deficient in two essential amino acids, lysine, and tryptophan. This deficiency results in an imbalance of amino acids and low protein content; normal maize varieties have less than half of the recommended amino acids for human nutrition. In addition, studies have shown that this deficiency has been associated with symptoms of growth impairment, anemia, hypoproteinemia, and fatty liver. Due to the fact that most of the presently available maize varieties do not contain the quality and quantity of proteins necessary for a balanced diet, different countries have focused on the research of quality protein maize (QPM). Researchers have characterized QPM noting that these varieties may contain between 70 to 100% more residues of the amino acids essential for animal and human nutrition, lysine, and tryptophan, than common corn. Several countries in Africa, Latin America, as well as China, have incorporated QPM in their agricultural development plan. Large parts of these countries have chosen a specific QPM variety based on their local needs and climate. Reviews have described the breeding methods of maize and have revealed the lack of studies on genetic and proteomic diversity of proteins in QPM varieties, and their genetic relationships with normal maize varieties. Therefore, molecular marker identification using tools such as mass spectrometry may accelerate the selection of plants that carry the desired proteins with high lysine and tryptophan concentration. To date, QPM maize lines have played a very important role in alleviating the malnutrition, and better characterization of these lines would provide a valuable nutritional enhancement for use in the resource-poor regions of the world. Thus, the objectives of this study were to identify proteins in QPM maize in comparison with a common maize line as a control.

Keywords: corn, mass spectrometry, QPM, tryptophan

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Authors: Yuting Wu, Faraz Choudhury, Daniel Benjamin, James Whalin, Joshua Blatz, Leon Shohet, Michael Sussman, Mark Richards

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Plasma-Induced Modification of Biomolecules (PLIMB) has been developed as a technology, which, together with mass spectrometry, measures three-dimensional structural characteristics of proteins. This technique uses hydroxyl radicals generated by atmospheric-pressure plasma discharge to react with the solvent-accessible side chains of protein in an aqueous solution. In this work, we investigate the three-dimensional structure of hemoglobin and myoglobin using PLIMB. Additional modifications to these proteins, such as oxidation, fragmentations, and conformational changes caused by PLIMB are also explored. These results show that PLIMB, coupled with mass spectrometry, is an effective way to determine solvent access to hemoproteins. Furthermore, we show that many factors, including pH and the electrical parameters used to generate the plasma, have a significant influence on solvent accessibility.

Keywords: plasma, hemoglobin, myoglobin, solvent access

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Authors: Alexander A. Osmolovskiy, Anastasia V. Orekhova, Daria M. Bednenko, Yelyzaveta Boiko

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Micromycetes from Aspergillus genus can produce proteases capable of promoting proteolysis of hemostasis proteins or, along with hydrolytic activity, to show the ability to convert proenzymes of this system activating them into an active form. At the same time, practical medicine needs specific activators for quantitation of the level of some plasma enzymes, especially protein C and factor X, the lack of which leads to the development of thromboembolic diseases. Thus, some micromycetes of the genus Aspergillus were screened for the ability to synthesize extracellular proteases with promising activity for designing anti-thrombotic and diagnostic preparations. Such standard methods like salting out, electrophoresis, isoelectrofocusing were used for isolation, purification and study of physicochemical properties of proteases. Enzyme activity was measured spectrophotometrically fibrin as a substrate of the reaction and chromogenic peptide substrates of different proteases of the human hemostasis system. As a result of the screening, four active producers were selected: Aspergillus janus 301, A. flavus 1, A. terreus 2, and A. ochraceus L-1. The enzyme of A. janus 301 showed the greatest fibrinolytic activity (around 329.2 μmol Tyr/(ml × min)). The protease produced by A. terreus 2 had the highest plasmin-like activity (54.1 nmol pNA/(ml × min)), but fibrinolytic activity was lower than A. janus 301 demonstrated (25.2 μmol Tyr/(ml × min)). For extracellular protease of micromycete A. flavus a high plasmin-like activity was also shown (39.8 nmol pNA / (ml × min)). Moreover, according to our results proteases one of the fungi - A. terreus 2 were able to activate protein C of human plasma - the key factor of the human anticoagulant hemostasis system. This type of activity was 39.8 nmol pNA/(ml × min)). It was also shown that A. ochraceus L-1 could produce extracellular proteases with protein C and factor X activator activities (65.9 nmol pNA/(ml × min) and 34.6 nmol pNA/(ml × min) respectively). The maximum accumulation of the proteases falls on the 4th day of cultivation. Using isoelectrofocusing was demonstrated that the activation of both proenzymes might proceed via limited proteolysis induced by proteases of A. ochraceus L-1. The activatory activity of A. ochraceus L-1 proteases toward essential hemostatic proenzymes, protein C and X factor may be useful for practical needs. It is well known that similar enzymes, activators of protein C and X factor isolated from snake venom, South American copperhead Agkistrodon contortrix contortrix and Russell’s viper Daboia russelli russeli, respectively, are used for the in vitro diagnostics of the functional state of these proteins in blood plasma. Thus, the proteases of Aspergillus genus can be used as cheap components for enzyme thrombolytic preparations.

Keywords: anti-trombotic drugs, fibrinolysis, diagnostics, proteases, micromycetes

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Authors: Akhil Raj Pushparajan, Ranjit Ramachandran, Jijimole Gopi Reji, Ajay Kumar Ramakrishnan

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death by an infectious disease. In spite of the availability of effective drugs and a vaccine, TB is a major health concern and was declared a global emergency by the World Health Organization (WHO). The success of intracellular pathogens like Mtb depends on its ability to overcome the challenging environment in the host. Gene regulation controlled by transcriptional regulators (TRs) plays a crucial role for the bacteria to adapt to the host environment. In vitro studies on gene regulatory mechanisms during dormancy and reactivation have provided insights into the adaptations employed by Mtb to survive in the host. Here we present our efforts to functionally characterize Rv1019, a putative TR of Mtb H37Rv which was found to be present at significantly varying levels during dormancy and reactivation in vitro. The expression of this protein in the dormancy-reactivation model was validated by qRT-PCR and western blot. By DNA- protein interaction studies and reporter assays we found that under normal laboratory conditions of growth this protein behaves as an auto-repressor and tetracycline was found to abrogate this repression by interfering with its ability to bind DNA. Further, by cDNA analysis, we found that this TR is co-transcribed with its downstream genes Rv1020 (mfd) and Rv1021 (mazG) which are involved in DNA damage response in Mtb. Constitutive expression of this regulator in the surrogate host M. smegmatis showed downregulation of the orthologues of downstream genes suggested that Rv1019 could negatively regulate these genes. Our finds also show that M. smegmatis expressing Rv1019 is sensitive to DNA damage suggests the role of this protein in regulating DNA damage response induced by oxidative stress. Because of its role in regulating DNA damage response which may help in the persistence of Mtb, Rv1019 could be used as a prospective target for therapeutic intervention to fight TB.

Keywords: auto-repressor, DNA repair, mycobacterium smegmatis, mycobacterium tuberculosis, tuberculosis

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Authors: Wael El-Deeb

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The present study aimed to investigate the diagnostic importance of acute phase proteins in urinary tract infection (UTI) in cattle. We describe the clinical, bacteriological and biochemical findings in 99 lactating cows. Blood and urine samples from diseased (n=84) and control healthy cows (n=15) were submitted to laboratory investigations. The urine analysis revealed hematuria and pyuria in UTI group. The isolated bacteria were E.coli (43/84) Corynebacterium spp, (31/84), Proteus spp. (6/84) and Streptococcus spp (4/84). The concentrations of Haptoglobin (Hp), serum amyloid A (SAA), α1-Acid glycoprotein (AGP), fibrinogen (Fb), total protein, albumen, and globulin were higher in cows with UTI when compared to healthy ones. Fifty-one of 84 cows with UTI were successfully treated. The levels of Hp, SAA, AGP, total protein, and globulin were associated with the odds of treatment failure. Conclusively, acute phase proteins could be used as diagnostic and prognostic biomarkers in cows with UTI.

Keywords: cows, urinary, infections, haptoglobin, serum Amyloid A

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