Search results for: milk protein

Authors: Awf A. Al-Khan, Michael J. Day, Judith Nimmo, Mourad Tayebi, Stewart D. Ryan, Samantha J. Richardson, Janine A. Danks

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Osteosarcoma (OS) is the most common type of malignant primary bone tumour in dogs. In addition to their critical roles in bone formation and remodeling, parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) are involved in progression and metastasis of many types of tumours in humans. The aims of this study were to determine the localisation and expression levels of PTHrP and PTHR1 in canine OS tissues using immunohistochemistry and to investigate if this expression is correlated with survival time. Formalin-fixed, paraffin-embedded tissue samples from 44 dogs with known survival time that had been diagnosed with primary osteosarcoma were analysed for localisation of PTHrP and PTHR1. Findings showed that both PTHrP and PTHR1 were present in all OS samples. The dogs with high level of PTHR1 protein (16%) had decreased survival time (P<0.05) compared to dogs with less PTHR1 protein. PTHrP levels did not correlate with survival time (P>0.05). The results of this study indicate that the PTHR1 is expressed differently in canine OS tissues and this may be correlated with poor prognosis. This may mean that PTHR1 may be useful as a prognostic indicator in canine OS and could represent a good therapeutic target in OS.

Keywords: dog, expression, osteosarcoma, parathyroid hormone receptor 1 (PTHR1), parathyroid hormone-related protein (PTHrP), survival

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Authors: Ibrahim Iddrisu, Joseph Ayariga, Junhuan Xu, Ayomide Adebanjo, Boakai K. Robertson, Michelle Samuel-Foo, Olufemi Ajayi

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The rise of antimicrobial resistance is a global public health crisis that threatens the effective control and prevention of infections. Due to the emergence of pan drug-resistant bacteria, most antibiotics have lost their efficacy. Bacteriophages or their components are known to target bacterial cell walls, cell membranes, and lipopolysaccharides (LPS) and hydrolyze them. Bacteriophages, being the natural predators of pathogenic bacteria, are inevitably categorized as ‘human friends’, thus fulfilling the adage that ‘the enemy of my enemy is my friend’. Leveraging on their lethal capabilities against pathogenic bacteria, researchers are searching for more ways to overcome the current antibiotic resistance challenge. In this study, we expressed and purified epsilon 34 phage tail spike protein (E34 TSP) from the E34 TSP gene, then assessed the ability of this bacteriophage protein in the killing of two CBD-resistant strains of Salmonella spp. We also assessed the ability of the tail spike protein to cause bacteria membrane disruption and dehydrogenase depletion. We observed that the combined treatment of CBD-resistant strains of Salmonella with CBD and E34 TSP showed poor killing ability, whereas the mono treatment with E34 TSP showed considerably higher killing efficiency. This study demonstrates that the inhibition of the bacteria by E34 TSP was due in part to membrane disruption and dehydrogenase inactivation by the protein. The results of this work provide an interesting background to highlight the crucial role phage proteins such as E34 TSP could play in pathogenic bacterial control.

Keywords: cannabidiol, resistance, Salmonella, antimicrobials, phages

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Authors: Eman. K. Ramadan, Abdelgawad. S. El-Tahawy

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The objective of this study was to evaluate the effects of calving season on the production and economic efficiency of dairy farms in Egypt. Our study was performed at dairy production farms in the Alexandria, Behera, and Kafr El-Sheikh provinces of Egypt from summer 2010 to winter 2013. The randomly selected dairy farms had herds consisting of Baladi, Holstein-Friesian, or cross-bred (Baladi × Holstein-Friesian) cows. The data were collected from production records and responses to a structured questionnaire. The average total return differed significantly (P < 0.05) between the different cattle breeds and calving seasons. The average total return was highest for the Holstein-Friesian cows that calved in the winter (29106.42 EGP/cow/year), and it was lowest for Baladi cows that calved in the summer (12489.79 EGP/cow/year). Differences in total returns between the cows that calved in the winter or summer or between the foreign and native breeds, as well as variations in calf prices, might have contributed to the differences in milk yield. The average net profit per cow differed significantly (P < 0.05) between the cattle breeds and calving seasons. The average net profit values for the Baladi cows that calved in the winter or summer were 2413 and 2994.96 EGP/cow/year, respectively, and those for the Holstein-Friesian cows were 10744.17 and 7860.56 EGP/cow/year, respectively, whereas those for the cross-bred cows were 10174.86 and 7571.33 EGP/cow/year, respectively. The variations in net profit might have resulted from variation in the availability or price of feed materials, milk prices, or sales volumes. Our results show that the breed and calving season of dairy cows significantly affected the economic efficiency of dairy farms in Egypt. The cows that calved in the winter produced more milk than those that calved in the summer, which may have been the result of seasonal influences, such as temperature, humidity, management practices, and the type of feed or green fodder available.

Keywords: calving season, economic, production, efficiency, dairy

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Authors: Chuanpeng Zhou

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The effect of dietary carbohydrate (CHO) level on serum physiological response, hepatic antioxidant abilities and heat shock protein 70 (HSP70) expression of Wuchang bream (Megalobrama amblycephala) was studied. Two isonitrogenous (28.56% crude protein) and isolipidic (5.28% crude lipid) diets were formulated to contain 30% or 53% wheat starch. Diets were fed for 90 days to fish in triplicate tanks (28 fish per tank). At the end of feeding trial, significantly higher serum triglyceride level, insulin level, cortisol level, malondialdehyde (MDA) content were observed in fish fed the 53% CHO diet, while significantly lower serum total protein content, alkaline phosphatase (AKP) activity, superoxide dismutase (SOD) activity and total antioxidative capacity (T-AOC) were found in fish fed the 53% CHO diet compared with those fed the 30% diet. The relative level of hepatic heat shock protein 70 mRNA was significantly higher in the 53% CHO group than that in the 30% CHO at 6, 12, and 48 h after feeding. The results of this study indicated that ingestion of 53% dietary CHO impacted the nonspecific immune ability and caused metabolic stress of Megalobrama amblycephala.

Keywords: Megalobrama amblycephala, carbohydrate, fasted and postprandial response, immunity, Hsp70

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Authors: Hamidreza Saberkari, Mousa Shamsi, Hossein Ahmadi, Saeed Vaali, , MohammadHossein Sedaaghi

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In the recent years, using signal processing tools for accurate identification of the protein coding regions has become a challenge in bioinformatics. Most of the genomic signal processing methods is based on the period-3 characteristics of the nucleoids in DNA strands and consequently, spectral analysis is applied to the numerical sequences of DNA to find the location of periodical components. In this paper, a novel ensemble algorithm for gene selection in DNA sequences has been presented which is based on the combination of Goertzel algorithm and anti-notch filter (ANF). The proposed algorithm has many advantages when compared to other conventional methods. Firstly, it leads to identify the coding protein regions more accurate due to using the Goertzel algorithm which is tuned at the desired frequency. Secondly, faster detection time is achieved. The proposed algorithm is applied on several genes, including genes available in databases BG570 and HMR195 and their results are compared to other methods based on the nucleotide level evaluation criteria. Implementation results show the excellent performance of the proposed algorithm in identifying protein coding regions, specifically in identification of small-scale gene areas.

Keywords: protein coding regions, period-3, anti-notch filter, Goertzel algorithm

Procedia PDF Downloads 388

Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

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PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provides better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

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Authors: Ghufran Ahmed, Muhammad Osama Siddiqui, Shahbaz Siddiqui, Rauf Ahmad Shams Malick, Faisal Khan, Mubashir Khan

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In this paper, a machine learning-based approach for early prediction of diseases in cows is proposed. Different ML algos are applied to extract useful patterns from the available dataset. Technology has changed today’s world in every aspect of life. Similarly, advanced technologies have been developed in livestock and dairy farming to monitor dairy cows in various aspects. Dairy cattle monitoring is crucial as it plays a significant role in milk production around the globe. Moreover, it has become necessary for farmers to adopt the latest early prediction technologies as the food demand is increasing with population growth. This highlight the importance of state-ofthe-art technologies in analyzing how important technology is in analyzing dairy cows’ activities. It is not easy to predict the activities of a large number of cows on the farm, so, the system has made it very convenient for the farmers., as it provides all the solutions under one roof. The cattle industry’s productivity is boosted as the early diagnosis of any disease on a cattle farm is detected and hence it is treated early. It is done on behalf of the machine learning output received. The learning models are already set which interpret the data collected in a centralized system. Basically, we will run different algorithms on behalf of the data set received to analyze milk quality, and track cows’ health, location, and safety. This deep learning algorithm draws patterns from the data, which makes it easier for farmers to study any animal’s behavioral changes. With the emergence of machine learning algorithms and the Internet of Things, accurate tracking of animals is possible as the rate of error is minimized. As a result, milk productivity is increased. IoT with ML capability has given a new phase to the cattle farming industry by increasing the yield in the most cost-effective and time-saving manner.

Keywords: IoT, machine learning, health care, dairy cows

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Authors: Arash Khorasani Esmaeili, Rosna Mat Taha, Sadegh Mohajer

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Seeds of Trifolium pratense (Red clover) were exposed in vitro for 6 weeks to six levels of lead (Pb) concentrations (0, 50, 100, 150, 200, 250 µM) to analyze the effects on growth, total chlorophyll and total protein contents of grown plants against the lead accumulation. The growth of plants was negatively affected by various levels of lead treatment. The fresh and dry weights, as well as lengths of shoots and roots of grown plants under various lead treatments, were found significantly lower in comparison with the control plants. Total chlorophyll and total soluble protein contents of grown plants under lower concentrations of lead treatment did not show significant differences when compared with the control plants, although they were affected significantly in higher levels of lead accumulation (150-250 µM).

Keywords: trifolium pratense, lead accumulation, chlorophyll content, protein content

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Authors: Min-Shiuan Liou, Yi Shan Yang, Yang-Zhan Huang, Chia-Wen Hsieh

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A high speed growing and butanol-tolerant Clostridium acetobutylicum HOL1 mutant was screened throughout continuous adaption culture with C. acetobutylicum ATCC 824. The HOL1 strain can grow well in 10 g/L butanol contained CGM medium and can produce about 12.8 g /L butanol during 24 hrs. The C. acetobutylicum HOL1 strain was able to produce 166 mM butanol with 21 mM acetone at pH 4.8, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.79, which is much higher than that (0.6) of the wild-type strain C. acetobutylicum ATCC 824. The acetate and butyrate accumulation were not observed during fermentation of the HOL1 strain. A hyper-butanol producing C. acetobutylicum HOL1 (pBPHS-3), which was created to overexpress the Bacillus psychrosaccharolyticus originated specific heat-shock protein gene, hspX, from a clostridial phosphotransbutyrylase promoter, was studied for its potential to produce a high titer of butanol. Overexpression of hspX resulted in increased final butanol yield 47% and 30% higher than those of the the ATCC824 and the HOL1 strains, respectively. The remarkable high-speed growth and butanol tolerance of strain HOL1 (pBPHS-3) demonstrates that overexpression of heterogeneous stress protein-encoding gene, hspX, could help C. acetobutylicum to effectively produce a high concentration of butanol.

Keywords: Clostridium acetobutylicum, butanol, heat-shock protein, resistance

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Authors: Jessica Pinheiro Silva, Brenda Rabello De Camargo, Daniel Gusmao De Morais, Eliane Ferreira Noronha

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The utilization of lignocellulosic biomass as source of polysaccharides for industrial applications requires an arsenal of enzymes with different mode of action able to hydrolyze its complex and recalcitrant structure. Clostridium thermocellum is gram-positive, thermophilic bacterium producing lignocellulosic hydrolyzing enzymes in the form of multi-enzyme complex, termed celulossomes. This complex has several hydrolytic enzymes attached to a large and enzymically inactive protein known as Cellulosome-integrating protein (CipA), which serves as a scaffolding protein for the complex produced. This attachment occurs through specific interactions between cohesin modules of CipA and dockerin modules in enzymes. The present work aims to construct celulosomes in vitro with the structural protein CipA, a xylanase called Xyn10D and a cellulose called CelJ from C.thermocellum. A mini-scafoldin was constructed from modules derived from CipA containing two cohesion modules. This was cloned and expressed in Escherichia coli. The other two genes were cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9 and integrated into the genome of the methylotrophic yeast Pichia pastoris GS115. Purification of each protein is being carried out. Further studies regarding enzymatic activity of the cellulosome is going to be evaluated. The cellulosome built in vitro and composed of mini-CipA, CelJ and Xyn10D, can be very interesting for application in industrial processes involving the degradation of plant biomass.

Keywords: cellulosome, CipA, Clostridium thermocellum, cohesin, dockerin, yeast

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Authors: Mohamed A. Morsy, Azza A. K. El-Sheikh, Abdulla Y. Al-Taher

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The aim of the present study is to investigate the effect of resveratrol (RES) on methotrexate (MTX)-induced testicular damage. RES (10 mg/kg/day) was given for 8 days orally and MTX (20 mg/kg i.p.) was given at day 4 of experiment, with or without RES in rats. MTX decreased serum testosterone, induced histopathological testicular damage, increased testicular tumor necrosis factor-α level and expression of nuclear factor-κB and cyclooxygenase-2. In MTX/RES group, significant reversal of these parameters was noticed, compared to MTX group. Testicular expression of multidrug resistance protein (Mrp) 3 was three- and five-folds higher in RES- and MTX/RES-treated groups, respectively. In vitro, using prostate cancer cells, each of MTX and RES alone induced cytotoxicity with IC50 0.18 ± 0.08 and 20.5 ± 3.6 µM, respectively. RES also significantly enhanced cytotoxicity of MTX. In conclusion, RES appears to have dual beneficial effect, as it promotes MTX tumor cytotoxicity, while protecting the testes, probably via up-regulation of testicular Mrp3 as a novel mechanism.

Keywords: resveratrol, methotrexate, multidrug resistance protein 3, tumor necrosis factor-α, nuclear factor-κB, cyclooxygenase-2

Procedia PDF Downloads 455

Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

Abstract:

PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provide better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

Procedia PDF Downloads 71

Authors: Mario Pérez-Won, Anais Palma-Acevedo, Luis González-Cavieres, Roberto Lemus-Mondaca, Gipsy Tabilo-Munizaga

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The Chilean abalone (Concholepas Concholepas) is a gastropod mollusk; it has a high commercial value due to the qualities of its meat, especially hardness, as a critical acceptance parameter. However, its main problem is its short shelf-life which is usually extended using traditional technologies with high energy consumption. Therefore, applying different technologies for the pre-treatment and drying process is necessary. In this research, pulsed electric field (PEF) was used as a pre-treatment for vacuum microwave drying (VMD), freeze-drying (FD), and hot-air drying (HAD). Drying conditions and characteristics were set according to previous experiments. The Drying samples were analyzed in terms of physical quality (color, texture, microstructure, and rehydration capacity), protein quality (degree of hydrolysis and computer protein efficiency ratio), and energy parameters. Regarding quality, the treatment that obtained lower harness was PEF+FD (195 N ± 10), the lowest change of color was for treatment PEF+VMD (ΔE: 17 ± 1.5), and the best rehydration capacity was for treatment PEF+VMD (1.2 h for equilibrium). For protein quality, the highest Computer-Protein Efficiency Ratio was the sample 2.0 kV/ cm of PEF (index of 4.18 ± 0.26 at the end of the digestion). Moreover, about energetic consumption, results show that VMD decreases the drying process by 97% whether PEF was used or not. Consequently, it is possible to conclude that using PEF as a pre-treatment for VMD and FD treatments has advantages that must be used following the consumer’s needs or preferences.

Keywords: chilean abalone, freeze-drying, proteins, pulsed electric fields

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Authors: Naureen Akhtar

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The stability of the protein in detergent-containing solution is the key for its successful crystallisation. Fluorescence-detection size-exclusion chromatography (FSEC) is a potential approach for screening monodispersity as well as the stability of protein in a detergent-containing-solution. In this present study, covalently linked Green Fluorescent Protein (GFP) to bacterial nitrate transporter, NarU from Escherichia coli was studied for pre-crystallisation trials by FSEC. Immobilised metal ion affinity chromatography (IMAC) and gel filtration were employed for their purification. The main objectives of this study were over-expression, detergent screening and crystallisation of nitrate transporter proteins. This study could not produce enough proteins that could realistically be taken forward to achieve the objectives set for this particular research. In future work, different combinations of variables like vectors, tags, creation of mutant proteins, host cells, position of GFP (N- or C-terminal) and/or membrane proteins would be tried to determine the best combination as the principle of technique is still promising.

Keywords: transporters, detergents, over-expression, crystallography

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Authors: M. Kowalski, M. Brodowski, K. Dziabowska, E. Czaczyk, W. Bialobrzeska, N. Malinowska, S. Zoledowska, R. Bogdanowicz, D. Nidzworski

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Boron-doped-carbon nanowall (BCNW) electrodes are recently in much interest among scientists. BCNWs are good candidates for biosensor purposes as they possess interesting electrochemical characteristics like a wide potential range and the low difference between redox peaks. Moreover, from technical parameters, they are mechanically resistant and very tough. The production process of the microwave plasma-enhanced chemical vapor deposition (MPECVD) allows boron to build into the structure of the diamond being formed. The effect is the formation of flat, long structures with sharp ends. The potential of these electrodes was checked in the biosensing field. The procedure of simple carbon electrodes modification by antibodies was adopted to BCNW for specific antigen recognition. Surface protein D deriving from H. influenzae pathogenic bacteria was chosen as a target analyte. The electrode was first modified with the aminobenzoic acid diazonium salt by electrografting (electrochemical reduction), next anti-protein D antibodies were linked via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry, and free sites were blocked by BSA. Cyclic voltammetry measurements confirmed the proper electrode modification. Electrochemical impedance spectroscopy records indicated protein detection. The sensor was proven to detect protein D in femtograms. This work was supported by the National Centre for Research and Development (NCBR) TECHMATSTRATEG 1/347324/12/NCBR/ 2017.

Keywords: anti-protein D antibodies, boron-doped carbon nanowall, impedance spectroscopy, Haemophilus influenzae.

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Authors: Roshni Raha, Karthikeyan S.

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The world's largest livestock population resides in India. Existing strategies must be modified to increase the production of livestock and their by-products in order to meet the demands of the growing human population. Even though India leads the world in both milk production and the number of cows, average production is not very healthy and productive. This may be due to the animals' poor nutrition caused by a chronic under-availability of high-quality fodder and feed. This article explores Azolla pinnata to be a promising source to produce high-quality unconventional feed and fodder for effective livestock production and good quality breeding in India. This article is an exploratory study using a literature survey and experimentation analysis. In the realm of agri-biotechnology, azolla sp gained attention for helping farmers achieve sustainability, having minimal land requirements, and serving as a feed element that doesn't compete with human food sources. It has high methionine content, which is a good source of protein. It can be easily digested as the lignin content is low. It has high antioxidants and vitamins like beta carotene, vitamin A, and vitamin B12. Using this concept, the paper aims to investigate and develop a model of using azolla plants as a novel, high-potential feed source to combat the problems of low production and poor quality of animals in India. A representative sample of animal feed is collected where azolla is added. The sample is ground into a fine powder using mortar. PITC (phenylisothiocyanate) is added to derivatize the amino acids. The sample is analyzed using HPLC (High-Performance Liquid Chromatography) to measure the amino acids and monitor the protein content of the sample feed. The amino acid measurements from HPLC are converted to milligrams per gram of protein using the method of amino acid profiling via a set of calculations. The amino acid profile data is then obtained to validate the proximate results of nutrient enhancement of the composition of azolla in the sample. Based on the proximate composition of azolla meal, the enhancement results shown were higher compared to the standard values of normal fodder supplements indicating the feed to be much richer and denser in nutrient supply. Thus azolla fed sample proved to be a promising source for animal fodder. This would in turn lead to higher production and a good breed of animals that would help to meet the economic demands of the growing Indian population. Azolla plants have no side effects and can be considered as safe and effective to be immersed in the animal feed. One area of future research could begin with the upstream scaling strategy of azolla plants in India. This could involve introducing several bioreactor types for its commercial production. Since azolla sp has been proved in this paper as a promising source for high quality animal feed and fodder, large scale production of azolla plants will help to make the process much quicker, more efficient and easily accessible. Labor expenses will also be reduced by employing bioreactors for large-scale manufacturing.

Keywords: azolla, fodder, nutrient, protein

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Authors: Caleb B. Sagwa, Tobias O. Okeno, Alexander K. Kahi

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Dairy cattle production plays an important role in the Kenyan economy. However, high incidences of mastitis is a major setback to the productivity in this industry. The current dairy cattle breeding objective in Kenya does not include mastitis resistance, mainly because the economic value of mastitis resistance has not been determined. Therefore this study aimed at estimating the economic value of mastitis resistance in dairy cattle in Kenya. Initial input parameters were obtained from literature on dairy cattle production systems in the tropics. Selection index methodology was used to derive the economic value of mastitis resistance. Somatic cell count (SCC) was used an indicator trait for mastitis resistance. The economic value was estimated relative to milk yield (MY). Economic values were assigned to SCC in a selection index such that the overall gain in the breeding goal trait was maximized. The option of estimating the economic value for SCC by equating the response in the trait of interest to its index response was considered. The economic value of mastitis resistance was US $23.64 while maximum response to selection for MY was US $66.01. The findings of this study provide vital information that is a pre-requisite for the inclusion of mastitis resistance in the current dairy cattle breeding goal in Kenya.

Keywords: somatic cell count, milk quality, payment system, breeding goal

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Authors: Zi-Han Li, Lu Fang, Liang Wu, Dong-Yuan Chang, Manyuan Dong, Liang Ji, Qi Zhang, Ming-Hui Zhao, Sydney C. W. Tang, Lemin Zheng, Min Chen

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Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for diabetic nephropathy (DN). Recently, we found that ANXA1 could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Here, we identified uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) by combining it with thioredoxin. Moreover, specific overexpression of UCP1 in the renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. This work established an alternative principle to harness the power of uncouplers for the treatment of DN.

Keywords: diabetic nephropathy, uncoupling protein 1, mitochondrial homeostasis, cardiolipin metabolism

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Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: tPA, recombinant, transgenic, tobacco

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Authors: Mohamad Ammar Ayass, Natalya Griko, Victor Pashkov, Wanying Cao, Kevin Zhu, Jin Zhang, Lina Abi Mosleh

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Respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19). Early evidence pointed at the angiotensin-converting enzyme 2 (ACE-2) expressed on the epithelial cells of the lung as the main entry point of SARS-CoV-2 into the cells. The viral entry is mediated by the binding of the Receptor Binding Domain (RBD) of the spike protein that is expressed on the surface of the virus to the ACE-2 receptor. As the number of SARS-CoV-2 variants continues to increase, mutations arising in the RBD of SARS-CoV-2 may lead to the ineffectiveness of RBD targeted neutralizing antibodies. To address this limitation, the objective of this study is to develop a combination of aptamers that target different regions of the RBD, preventing the binding of the spike protein to ACE-2 receptor and subsequent viral entry and replication. A safe and innovative biomedical tool was developed to inhibit viral infection and reduce the harms of COVID-19. In the present study, DNA aptamers were developed against a recombinant trimer S protein using the Systematic Evolution of Ligands by Exponential enrichment (SELEX). Negative selection was introduced at round number 7 to select for aptamers that bind specifically to the RBD domain. A series of 9 aptamers (ADI2010, ADI2011, ADI201L, ADI203L, ADI205L, ADIR68, ADIR74, ADIR80, ADIR83) were selected and characterized with high binding affinity and specificity to the RBD of the spike protein. Aptamers (ADI25, ADI2009, ADI203L) were able to bind and pull down endogenous spike protein expressed on the surface of SARS-CoV-2 virus in COVID-19 positive patient samples and determined by liquid chromatography- tandem mass spectrometry analysis (LC-MS/MS). LC-MS/MS data confirmed that aptamers can bind to the RBD of the spike protein. Furthermore, results indicated that the combination of the 9 best aptamers inhibited the binding of the purified trimer spike protein to the ACE-2 receptor found on the surface of Vero E6 cells. In the same experiment, the combined aptamers displayed a better neutralizing effect than antibodies. The data suggests that the selected aptamers could be used in therapy to neutralize the effect of the SARS-CoV-2 virus by inhibiting the interaction between the RBD and ACE-2 receptor, preventing viral entry into target cells and therefore blocking viral replication.

Keywords: aptamer, ACE-2 receptor, binding inhibitor, COVID-19, spike protein, SARS-CoV-2, treatment

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Authors: Ç. Gökçek-Saraç, Ş. Özen, N. Derin

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The N-methyl-D-aspartate (NMDA)-dependent pathway is the major intracellular signaling pathway implemented in both short- and long-term memory formation in the hippocampus which is the most studied brain structure because of its well documented role in learning and memory. However, little is known about the effects of RF-EMR exposure on NMDA receptor signaling pathway including activation of protein kinases, notably Ca²⁺/calmodulin-dependent protein kinase II alpha (CaMKIIα). The aim of the present study was to investigate the effects of acute and chronic 900 MHz RF-EMR exposure on both passive avoidance behaviour and hippocampal levels of CaMKIIα and its phosphorylated form (pCaMKIIα). Rats were divided into the following groups: Sham rats, and rats exposed to 900 MHz RF-EMR for 2 h/day for 1 week (acute group) or 10 weeks (chronic group), respectively. Passive avoidance task was used as a behavioural method. The hippocampal levels of selected kinases were measured using Western Blotting technique. The results of passive avoidance task showed that both acute and chronic exposure to 900 MHz RF-EMR can impair passive avoidance behaviour with minor effects on chronic group of rats. The analysis of western blot data of selected protein kinases demonstrated that hippocampal levels of CaMKIIα and pCaMKIIα were significantly higher in chronic group of rats as compared to acute groups. Taken together, these findings demonstrated that different duration times (1 week vs 10 weeks) of 900 MHz RF-EMR exposure have different effects on both passive avoidance behaviour of rats and hippocampal levels of selected protein kinases.

Keywords: hippocampus, protein kinase, rat, RF-EMR

Procedia PDF Downloads 255

Authors: Nasser A. Al-Shabib

Abstract:

Food allergens are most common non-native form when exposed to the immune system. Most food proteins undergo various treatments (e.g. thermal or proteolytic processing) during food manufacturing. Such treatments have the potential to impact the chemical structure of food allergens so as to convert them to more denatured or unfolded forms. The conformational changes in the proteins may affect the allergenicity of treated-allergens. However, most allergenic proteins possess high resistance against thermal modification or digestive enzymes. In the present study, ovomucoid (a major allergenic protein of egg white) was heated in phosphate-buffered saline (pH 7.4) at different temperatures, aqueous solutions and on different surfaces for various times. The results indicated that different antibody-based methods had different sensitivities in detecting the heated ovomucoid. When using one particular immunoassay‚ the immunoreactivity of ovomucoid increased rapidly after heating in water whereas immunoreactivity declined after heating in alkaline buffer (pH 10). Ovomucoid appeared more immunoreactive when dissolved in PBS (pH 7.4) and heated on a stainless steel surface. To the best of our knowledge‚ this is the first time that antibody-based methods have been applied for the detection of ovomucoid adsorbed onto different surfaces under various conditions. The results obtained suggest that use of antibodies to detect ovomucoid after food processing may be problematic. False assurance will be given with the use of inappropriate‚ non-validated immunoassays such as those available commercially as ‘Swab’ tests. A greater understanding of antibody-protein interaction after processing of a protein is required.

Keywords: ovomucoid, thermal treatment, solutions, surfaces

Procedia PDF Downloads 449

Authors: Stéfani T. A. Dantas, Laura T. S. Takume, Bruna F. Rossi, Érika R. Bonsaglia, Ivana G. Castilho, José C. F. Pantoja, Ary Fernandes Júnior, Juliano L. Gonçalves, Marcos V. Santos, Rinaldo A. Mota, Vera L. M. Rall

Abstract:

Staphylococcus aureus is one of the main pathogens causing contagious bovine mastitis, being more frequently isolated from subclinical form, although the clinical form also occurs. Clinical mastitis cause visual signs, such as swelling, fever, hardening of the mammary gland, or any change in the characteristics of the milk. Considering the subclinical type, there are no visible signs in the animal nor changes in the milk. S. aureus has many important virulence factors for the establishment of its pathogenicity in animals, such as enterotoxins, which are also responsible for foodborne poisoning. Our objective is to perform a comparative analysis between 103 isolates of S. aureus, obtained from the milk of cows with clinical mastitis and 103 more, from subclinical type, in relation to the presence of these enterotoxins and verify if their presence plays an important role in the signs of illness. We will investigate all enterotoxins described till now, such as sea-see, seg-sez, sel26, sel 27, se01, and se02 (This study was approved by the Sao Paulo State University Animal Use Ethics Committee, No. 0136/2017). For the PCR assay, we used Illustra Bacteria Mini Spin Kit for bacterial DNA. At this moment, we have already tested sea-see, seg-ser, sew, and sex, and the results have already been submitted to Fisher Exact Probability Test or Chi-square Test. Considering the isolates obtained from clinical mastitis, the most frequent enterotoxins were selw (99%), selx (78%) and selh (50.5%), and sec, see, sej, sell, selp,and ser were absent. Among the subclinics, selw (82.5%) selm (15.5%) and selx (14.6%) were the most frequent, and sea-see, seg, sei-sel, sem-ser were absent. We have already observed statistically significant differences for seb, seg, seh, sei, selo, selu, selw and selx. Other interesting results were the low number of genes in each isolate from subclinical mastitis [0 genes: 14 (13.6%); 1 gene: 55 (53.4%); 2 genes: 33 (32%) or 3: 1 (0.97%)] compared to clinical isolates [1 gene: 5 (4.9%); 2 genes: 29 (28.1%); 3 genes: 38 (36.9%); 4 genes: 14 (13.6%); 5 genes: 5 (4.9%); 6 genes: 4 (3.9%); 7 genes: 5 (4.9%); 8 genes: 2 (1.9%) and 9 genes: 1 (1%)]. Based on these results, we can conclude that enterotoxins indeed play an important role in clinical signs in cattle with mastitis.

Keywords: mastitis, S. aureus, PCR, staphylococcal enterotoxin

Procedia PDF Downloads 114

Authors: Han-Bin Kim, Sooim Shin, Moonsung Choi

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There is a growing interest in introducing a desired functional group on enzymes in the field of protein engineering. In here, various redox centers were newly created using histidine tag, which is widely used for protein purification, plus cobalt in one of cupredoxins, amicyanin. The coordination of Cobalt-His tag and reactivity of the Co²⁺ loaded His-tag also were characterized. 3xHis-tag, 6xHis-tag, and 9xHis-tag were introduced on amicyanin by site-directed mutagenesis, and then Co²⁺ was loaded on each His-tagged amicyanin. The spectral changes at 330 nm corresponding to cobalt binding on His-tag site indicated the binding ratio of 3xHis-tag, 6xHis-tag, and 9xHis-tag to cobalt as 1:1, 1:2, 1:3 respectively. Based on kinetic studies of binding cobalt to 3xHis-tag, 6xHis-tag, and 9xHis-tagged amicyanin, the nature of the sites was elucidated. In addition, internal electron transfer properties between Cu¹⁺ site and engineered site of amicyanin were determined. These results provide insight into improvement of metal coordination and alternation of the redox properties of metal as a new catalytic site on proteins.

Keywords: amicyanin, cobalt, histidine, protein engineering

Procedia PDF Downloads 163

Authors: Ibinabo I. Ilaboya, Eustace A. Iyayi

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Phosphorus (P) is an indispensable mineral in broiler diets. Rice husk contains phytate-P and other nutrients like protein, carbohydrates, which are poorly digested in broiler chickens. Broiler chickens (BC) lacks sufficient phytase to help hydrolyse phytate-bound P. Hence excess of P is excreted by these chickens into the environment causing environmental pollution. Supplementation of such diets with microbial phytase helps to improve the digestibility of these nutrients. The study was conducted to determine the effect of phytase supplementation on the digestibility of crude protein (CP) and P of rice husk in BC. Six semi-purified diets of three levels of total P (3.46, 4.91 and 6.37g/kg) without and with 1,000 units of phytase per kg were formulated. Titanium dioxide was added to the diets at the rate of 5g/kg as an indigestible marker. At 20dposthatch, 288 broilers (Abor Acre) were weighed and allotted to the diets with 6 replicates of 8 birds each in a randomized complete block design. The birds had free access to the experimental diets until day 26 post-hatch. Phytase supplementation increased (p < 0.05) digestibility of P from 75-93%. Rice husk and its interaction with phytase had no significant (p > 0.05) effect on P digestibility, whereas there was significant (p < 0.01) effect on the interaction of rice husk with phytase on CP digestibility. There were linear increases (p < 0.01) in digested P and CP with phytase supplementation. The P and CP losses from the BC was reduced with the addition of phytase. Results suggest that supplementation of rice husk-based diets with microbial phytase improved pre-caecal digestibility of P and CP in broilers.

Keywords: crude protein, phosphorus, phytase, rice husk

Procedia PDF Downloads 143

Authors: Akhmadjon Sultanov, Eun-Ho Lee, Hye-Jin Park, Young-Je Cho

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This study tested the anti-inflammatory effect of wild indigo (Baptisia tinctoria) root in Raw 264.7 cells. We prepared two extracts of B. tinctoria; one in water and the other in 50% ethanol. Then we evaluated the toxicities of the B. tinctoria root extracts at 10 to 100 mg mL-1 concentrations in raw 264.7 cells and observed 80% cell viability. The anti-inflammatory effect of B. tinctoria root extract in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells were observed with concentrations at 10, 30, and 50 μg mL-1. The results showed that 77.27-66.82% of nitric oxide (NO) production was inhibited by 50 μg mL-1 B. tinctoria root extract. The protein expression of Inducible NO synthase (iNOS) expression dramatically decreased by 93.14% and 52.65% in raw 264.7 cells treated with water and ethanol extracts of B. tinctoria root, respectively. Moreover, cyclooxygenase-2 (COX-2) protein expression decreased by 42.85% and 69.70% in raw 264.7 cells treated with water and ethanol extracts of B. tinctoria root, respectively. Furthermore, the mRNA expression of pro-inflammatory markers, such as tumor necrosis factor-alpha, interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, and prostaglandin E synthase 2, was significantly suppressed in a concentration-dependent manner. Additionally, the B. tinctoria root extracts effectively inhibited enzymes involved in physiological activities. The B. tinctoria root extracts showed excellent anti-inflammatory effects and can be used as a functional material for biological activities.

Keywords: cytokine, macrophage, pro-inflammatory, protein expression, real-time PCR

Procedia PDF Downloads 71

Authors: M. Hetmańczyk, R. Polaniak, K. Brukało, E. Grochowska-Niedworok

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Eating behaviours of people are determined by knowledge gained at different stages of life. Children’s diet is especially important. They have to eat meals regularly. Meals should consist of protein, carbohydrates and fat, and drinking the right amount of water. Mistakes in children’s diets affect their health and may lead to health issues such as diabetes, overweight, obesity or malnutrition. The aim of the study was to assess the eating habits among 10-15-year-old children. To achieve this aim, the study included children aged 10-15 years living in Silesia Province, Poland; the participants consisted of 52.08% girls and 47.92% boys. Authorial questionnaire contains 28 questions about eating habits. The results of 192 students were subjected to analysis. The results show that half of the surveyed students participated in physical activity every day. Most children ate 4-5 meals every day, but the breaks between them were too long (four and more hours). Children generally ate cooked meals. Most children ate first breakfast every day, but only one third of studied children ate a second breakfast daily, while 93.75% ate vegetables at least once a day, 94.79% ate fruit at least once a day, and 79.17% drink a daily glass of milk or more. The study found that the eating behaviours of the surveyed children were unsatisfying. While the children did not participate in physical activity often enough, girls took part slightly more often. Children eat second breakfast not often enough. Younger children (10-12 years old) are doing it more often than the older children (13-15 years old). Gender is not a determinant of the frequency of second breakfast consumption.

Keywords: eating habits, children, diet, nutrition status

Procedia PDF Downloads 174

Authors: Zuzana Tatarkova, Ivana Pilchova, Michal Cibulka, Martin Kolisek, Peter Racay, Peter Kaplan

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Introduction: Normal functioning of mitochondria is crucial for cardiac performance. Mitochondria undergo mitophagy and biogenesis, and mitochondrial proteins are subject to extensive post-translational modifications. The state of mitochondrial homeostasis reflects overall cellular fitness and longevity. Perturbed mitochondria produce less ATP, release greater amounts of reactive molecules, and are more prone to apoptosis. Therefore mitochondrial turnover is an integral aspect of quality control in which dysfunctional mitochondria are selectively eliminated through mitophagy. Currently, the progressive deterioration of physiological functions is seen as accumulation of modified/damaged proteins with limiting regenerative ability and disturbance of such affected protein-protein communication throughout aging in myocardial cells. Methodologies: For our study was used immunohistochemistry, biochemical methods: spectrophotometry, western blotting, immunodetection as well as more sophisticated 2D electrophoresis and mass spectrometry for evaluation protein-protein interactions and specific post-translational modification. Results and Discussion: Mitochondrial stress response to reactive species was evaluated as electron transport chain (ETC) complexes, redox-active molecules, and their possible communication. Protein-protein interactions revealed a strong linkage between age and ETC protein subunits. Redox state was strongly affected in senescent mitochondria with shift in favor of more pro-oxidizing condition within cardiomyocytes. Acute myocardial ischemia and ischemia-reperfusion (IR) injury affected ETC complexes I, II and IV with no change in complex III. Ischemia induced decrease in total antioxidant capacity, MnSOD, GSH and catalase activity with recovery in some extent during reperfusion. While MnSOD protein content was higher in IR group, activity returned to 95% of control. Nitric oxide is one of the biological molecules that can out compete MnSOD for superoxide and produce peroxynitrite. This process is faster than dismutation and led to the 10-fold higher production of nitrotyrosine after IR injury in adult with higher protection in senescent ones. 2D protein profiling revealed 140 mitochondrial proteins, 12 of them with significant changes after IR injury and 36 individual nitrotyrosine-modified proteins further identified by mass spectrometry. Linking these two groups, 5 proteins were altered after IR as well as nitrated, but only one showed massive nitration per lowering content of protein after IR injury in adult. Conclusions: Senescent cells have greater proportion of protein content, which might be modulated by several post-translational modifications. If these protein modifications are connected to functional consequences and protein-protein interactions are revealed, link may lead to the solution. Assume all together, dysfunctional proteostasis can play a causative role and restoration of protein homeostasis machinery is protective against aging and possibly age-related disorders. This work was supported by the project VEGA 1/0018/18 and by project 'Competence Center for Research and Development in the field of Diagnostics and Therapy of Oncological diseases', ITMS: 26220220153, co-financed from EU sources.

Keywords: aging heart, mitochondria, proteomics, redox state

Procedia PDF Downloads 169

Authors: Yuexin Liu, Gordon B. Mills, Russell R. Broaddus, John N. Weinstein

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The lethality of endometrioid endometrial cancer (EEC) is primarily attributable to the high-stage diseases. However, there are no available biomarkers that predict EEC patient staging at the time of diagnosis. We aim to develop a predictive scheme to help in this regards. Using reverse-phase protein array expression profiles for 210 EEC cases from The Cancer Genome Atlas (TCGA), we constructed a Protein Scoring of EEC Staging (PSES) scheme for surgical stage prediction. We validated and evaluated its diagnostic potential in an independent cohort of 184 EEC cases obtained at MD Anderson Cancer Center (MDACC) using receiver operating characteristic curve analyses. Kaplan-Meier survival analysis was used to examine the association of PSES score with patient outcome, and Ingenuity pathway analysis was used to identify relevant signaling pathways. Two-sided statistical tests were used. PSES robustly distinguished high- from low-stage tumors in the TCGA cohort (area under the ROC curve [AUC]=0.74; 95% confidence interval [CI], 0.68 to 0.82) and in the validation cohort (AUC=0.67; 95% CI, 0.58 to 0.76). Even among grade 1 or 2 tumors, PSES was significantly higher in high- than in low-stage tumors in both the TCGA (P = 0.005) and MDACC (P = 0.006) cohorts. Patients with positive PSES score had significantly shorter progression-free survival than those with negative PSES in the TCGA (hazard ratio [HR], 2.033; 95% CI, 1.031 to 3.809; P = 0.04) and validation (HR, 3.306; 95% CI, 1.836 to 9.436; P = 0.0007) cohorts. The ErbB signaling pathway was most significantly enriched in the PSES proteins and downregulated in high-stage tumors. PSES may provide clinically useful prediction of high-risk tumors and offer new insights into tumor biology in EEC.

Keywords: endometrial carcinoma, protein, protein scoring of EEC staging (PSES), stage

Procedia PDF Downloads 222

Authors: E. Oluwasola, E. Bamidele, E. Ogunbusola

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Enriched “aadun” was produced from maize with, supplemented with cray fish and beans. Sodium chloride (Nacl) was also added to the product which acts as preservatives. The produced enriched “aadun” was compared with commercial “aadun” organoleptically the result of the sensory evaluation carried out on the product showed that there is a statistical significant difference between the mouth feel of enriched and commercial “aadun” at 0.05 level of significance (t=5.499, P<0.05) Similarly, the mean difference between enriched and commercial “aadun” in terms of aroma (t=4.403, P<0.05), taste (t=4.592, P<0.05) colour (t=2.788, P<0.05) and general acceptability (t=3.894, P<0.05) is statistically significant at 95% confidence level in each case, therefore, it is clearly revealed that product 321 (Enriched “aadun”) is more acceptable and significant better than product 432 (commercial “aadun”) in all the attributes evaluated. The proximate analysis using standard methods of analysis was carried out which include the moisture content, ash and protein content for both the enriched aadun and commercial aadun the result showed moisture content 9%, ash 6.2%, protein 19.6% and 12.9% moisture content, 4%ash content, 8.75% protein for the commercial and improved aadun respectively.

Keywords: aadun, enriched, maize, supplemented

Procedia PDF Downloads 557