Search results for: alternative proteins

Authors: Arash Jafari, Somaye Bahrami, Mohammad Hossein Razi Jalali

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The aim of this study was the isolation and identification of excretory-secretory and somatic antigens from D. dendriticum by SDS-PAGE and evaluation of humeral immune response against these antigens. The sera of infected sheep with different infection degrees were collected. Somatic and ES proteins were isolated with SDS PAGE. Immunogenicity properties of the resulting proteins were determined using western blot analysis. The total extract of somatic antigens analysed by SDS-PAGE revealed 21 proteins. In mild infection, bands of 130 KDa were immune dominant. In moderate infections 48, 80 and 130 KDa and in heavy infections 48, 60, 80, 130 KDa were detected as immune dominant bands. In ES antigens, mild infection 130 KDa, in moderate infection 100, 120 and 130 KDa and in heavy infection 45, 80, 85, 100, 120 and 130 KDa were immune dominant bands. The most immunogenic protein band during different degrees of infection was 130KDa.

Keywords: Dicrocoelium dendriticum excretory-secretory antigens, somatic antigens, western blot

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Authors: Deepak Bhardwaj, Narendra Tuteja

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Global climate change is impacting large agrarian societies, especially those in countries located near the equator. Agriculture, and consequently, plant-based food, is the hardest hit in tropical and sub-tropical countries such as India due to an increased incidence of drought as well as an increase in soil salinity. One method that holds promise is AMF-rich biofertilizers which assist in activating proteins which in turn help alleviate abiotic stress in plants. In the present study, we identified two important species of (arbuscular mycorrhizal fungus) AMF belonging to Glomus and Gigaspora from the rhizosphere of the important medicinal plant Justicia adathoda. These two species have been found to be responsible for the abundance of Justicia adathoda in the semi-arid areas of the Jammu valley located in northern India, namely, the Union Territory of Jammu and Kashmir. We isolated the species of Glomus and Gigaspora from the rhizosphere of Justicia adathoda and used them as biofertilizers for the tomato plant. Significant improvements in the growth parameters were observed in the tomato plants inoculated with Glomus sp. and Gigaspora sp. in comparison with the tomato plants that were grown without AMF treatments. Tomato plants grown along with Glomus sp. and Gigaspora sp. have been observed to withstand 200 mM of salinity and 25% PEG stress. AMF also resulted in an increased concentration of proline and antioxidant enzymes in tomato plants. We also examined the expression levels of salinity and drought stress-inducible genes such as pea DNA helicase 45 (PDH 45) and genes of G-protein subunits of the tomato plants inoculated with and without AMF under stress and normal conditions. All the stress-inducible genes showed a significant increase in their gene expression under stress and AMF inoculation, while their levels were found to be normal under AMF inoculation without stress. We propose a model of abiotic stress alleviation in tomato plants with the help of external factors such as AMF and internally with the help of proteins like PDH 45 and G-proteins.

Keywords: AMF, abiotic stress, g-proteins, PDH-45

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Authors: Hui-Ling Huang, Tamara Vasylenko, Phasit Charoenkwan, Shih-Hsiang Chiu, Shinn-Ying Ho

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The knowledge of the human transporters is still limited due to technically demanding procedure of crystallization for the structural characterization of transporters by spectroscopic methods. It is desirable to develop bioinformatics tools for effective analysis of available sequences in order to identify human transmembrane transporter proteins (HMTPs). This study proposes a scoring card method (SCM) based method for predicting HMTPs. We estimated a set of propensity scores of dipeptides to be HMTPs using SCM from the training dataset (HTS732) consisting of 366 HMTPs and 366 non-HMTPs. SCM using the estimated propensity scores of 20 amino acids and 400 dipeptides -as HMTPs, has a training accuracy of 87.63% and a test accuracy of 66.46%. The five top-ranked dipeptides include LD, NV, LI, KY, and MN with scores 996, 992, 989, 987, and 985, respectively. Five amino acids with the highest propensity scores are Ile, Phe, Met, Gly, and Leu, that hydrophobic residues are mostly highly-scored. Furthermore, obtained propensity scores were used to analyze physicochemical properties of human transporters.

Keywords: dipeptide composition, physicochemical property, human transmembrane transporter proteins, human transmembrane transporters binding propensity, scoring card method

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Authors: Abdollah Sobhani, Hossein Vaseghi-Dodaran

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Proteomics is defined as the study of the component of a cell, tissue and biological fluid. This technique has the potential to identify protein biomarkers of a disease states. In this study which was performed on bovine ovarian follicular cysts (BOFC), eight proteins are over expressed in BOFC that these proteins could be useful biomarkers for BOFC. The difference between serum proteome pattern cows affected by postpartum endometritis with healthy cows revealed that concentrations orosomucoid was decreased in endometritis. The comparison proteome of brucella abortus between laboratory adapted strains and clinical isolates could be useful to better understand this disease and vaccine development. Proteomics experiments identified new proteins and pathways that may be important in future hypothesis-driven studies of glucocorticoid-induced immunosuppression. Understanding the molecular mechanisms of effective parameters on male fertility is essential for obtaining high reproductive efficiency by decreasing cost and time. The investigations on proteome of high fertility spermatozoa indicated that expression of some proteins such as casein kinase 2 (CKII) prime poly peptide and tyrosine kinase in high fertility spermatozoa was higher compared to low fertility spermatozoa. Also, some evidence has indicated that variation in protein types and amounts in seminal fluid regulates fertility indexes in dairy bull. In conclusion, proteomics is a useful technique for discovering drugs, vaccine development, and diagnosis disease by biomarkers and improvement of reproduction efficiency.

Keywords: proteomics, reproduction, biomarker, immunity

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Authors: Patarasuda Chaisupa, R. Clay Wright

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The selection of appropriate biomolecular targets is a crucial aspect of biopharmaceutical development. The Auxin-Inducible Degron Degradation (AID) technology has demonstrated remarkable potential in efficiently and rapidly degrading target proteins, thereby enabling the identification and acquisition of drug targets. The AID system also offers a viable method to deplete specific proteins, particularly in cases where the degradation pathway has not been exploited or when the adaptation of proteins, including the cell environment, occurs to compensate for the mutation or gene knockout. In this study, we have engineered an improved AID system tailored to deplete proteins of interest. This AID construct combines the auxin-responsive E3 ubiquitin ligase binding domain, AFB2, and the substrate degron, IAA17, fused to the target genes. Essential genes of fungi with the lowest percent amino acid similarity to human and plant orthologs, according to the Basic Local Alignment Search Tool (BLAST), were cloned into the AID construct in S. cerevisiae (AID-tagged strains) using a modular yeast cloning toolkit for multipart assembly and direct genetic modification. Each E3 ubiquitin ligase and IAA17 degron was fused to a fluorescence protein, allowing for real-time monitoring of protein levels in response to different auxin doses via cytometry. Our AID system exhibited high sensitivity, with an EC50 value of 0.040 µM (SE = 0.016) for AFB2, enabling the specific promotion of IAA17::target protein degradation. Furthermore, we demonstrate how this improved AID system enhances quantitative functional studies of various proteins in fungi. The advancements made in auxin-inducible protein degradation in this study offer a powerful approach to investigating critical target protein viability in fungi, screening protein targets for drugs, and regulating intracellular protein abundance, thus revolutionizing the study of protein function underlying a diverse range of biological processes.

Keywords: synthetic biology, bioengineering, molecular biology, biotechnology

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Authors: Kiseob Chung, Hyeonmin Kang

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This study aims to explore what is intercultural competency needed for teacher through their experience at the public multicultural alternative school. The public alternative multicultural school is an accredited school for immigrants or students from multicultural families who have experienced school maladjustment at public school. This school has self-regulation in curriculum and function of bridge to public school by helping their adaptation. In particular, this study answers the following questions: What are the most difficulties for teacher at the multicultural alternative school in comparison to public school? What competencies are required for teacher at the multicultural alternative school? Which competencies in cognitive, emotional and practical area should be more required in order for teacher to communicate with student effectively (successfully) in class and other activities in school? What is the background of that we called these competencies especially as ‘intercultural’? This study focuses to clarify teacher’s competency to help immigrants of students from multicultural background to adjust to school life with the term of intercultural competency.

Keywords: intercultural competency for teacher, multicultural alternative school, multicultural students, school maladjustment

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Authors: Anuschka Curran, Sandra Barnard

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Coral reefs are of the most diverse and productive ecosystems on the planet, but due to the impact of climate change, these infrastructures are dying off primarily through coral bleaching. Coral bleaching can be described as the process by which zooxanthellae (algal endosymbionts) are expelled from the gastrodermal cavity of the respective coral host, causing increased coral whitening. The general consensus is that mass coral bleaching is due to the dysfunction of photosynthetic processes in the zooxanthellae as a result of the combined action of elevated temperature and light-stress. The question then is, do zooxanthellae have the potential to play a key role in the future of coral reef restoration through genetic engineering? The aim of this study is firstly to review the different zooxanthellae taxa and their traits with respect to environmental stress, and secondly, to review the information available on the protective mechanisms present in zooxanthellae cells when experiencing temperature fluctuations, specifically concentrating on heat shock proteins and the antioxidant stress response of zooxanthellae. The eight clades (A-H) previously recognized were redefined into seven genera. Different zooxanthellae taxa exhibit different traits, such as their photosynthetic stress responses to light and temperature. Zooxanthellae have the ability to determine the amount and type of heat shock proteins (hsps) present during a heat response. The zooxanthellae can regulate both the host’s respective hsps as well as their own. Hsps, generally found in genotype C3 zooxanthellae, such as Hsp70 and Hsp90, contribute to the thermal stress response of the respective coral host. Antioxidant activity found both within exposed coral tissue, and the zooxanthellae cells can prevent coral hosts from expelling their endosymbionts. The up-regulation of gene expression, which may mitigate thermal stress induction of any of the physiological aspects discussed, can ensure stable coral-zooxanthellae symbiosis in the future. It presents a viable alternative strategy to preserve reefs amidst climate change. In conclusion, despite their unusual molecular design, genetic engineering poses as a useful tool in understanding and manipulating variables and systems within zooxanthellae and therefore presents a solution that can ensure stable coral-zooxanthellae symbiosis in the future.

Keywords: antioxidant enzymes, genetic engineering, heat-shock proteins, Symbiodinium

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Authors: Olga V. Chervyakova, Elmira T. Tailakova, Vitaliy M. Strochkov, Kulyaisan T. Sultankulova, Nurlan T. Sandybayev, Lev G. Nemchinov, Rosemarie W. Hammond

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Sheep pox is a highly contagious infection that OIE regards to be one of the most dangerous animal diseases. It causes enormous economic losses because of death and slaughter of infected animals, lower productivity, cost of veterinary and sanitary as well as quarantine measures. To control spread of sheep pox infection the attenuated vaccines are widely used in the Republic of Kazakhstan and other Former Soviet Union countries. In spite of high efficiency of live vaccines, the possible presence of the residual virulence, potential genetic instability restricts their use in disease-free areas that leads to necessity to exploit new approaches in vaccine development involving recombinant DNA technology. Vaccines on the basis of recombinant proteins are the newest generation of prophylactic preparations. The main advantage of these vaccines is their low reactogenicity and this fact makes them widely used in medical and veterinary practice for vaccination of humans and farm animals. The objective of the study is to produce recombinant immunogenic proteins for development of the high-performance means for sheep pox prophylaxis. The SPV proteins were chosen for their homology with the known immunogenic vaccinia virus proteins. Assay of nucleotide and amino acid sequences of the target SPV protein genes. It has been shown that four proteins SPPV060 (ortholog L1), SPPV074 (ortholog H3), SPPV122 (ortholog A33) and SPPV141 (ortholog B5) possess transmembrane domains at N- or C-terminus while in amino acid sequences of SPPV095 (ortholog А 4) and SPPV117 (ortholog А 27) proteins these domains were absent. On the basis of these findings the primers were constructed. Target genes were amplified and subsequently cloned into the expression vector рЕТ26b(+) or рЕТ28b(+). Six constructions (pSPPV060ΔТМ, pSPPV074ΔТМ, pSPPV095, pSPPV117, pSPPV122ΔТМ and pSPPV141ΔТМ) were obtained for expression of the SPV genes under control of T7 promoter in Escherichia coli. To purify and detect recombinant proteins the amino acid sequences were modified by adding six histidine molecules at C-terminus. Induction of gene expression by IPTG was resulted in production of the proteins with molecular weights corresponding to the estimated values for SPPV060, SPPV074, SPPV095, SPPV117, SPPV122 and SPPV141, i.e. 22, 30, 20, 19, 17 and 22 kDa respectively. Optimal protocol of expression for each gene that ensures high yield of the recombinant protein was identified. Assay of cellular lysates by western blotting confirmed expression of the target proteins. Recombinant proteins bind specifically with antibodies to polyhistidine. Moreover all produced proteins are specifically recognized by the serum from experimentally SPV-infected sheep. The recombinant proteins SPPV060, SPPV074, SPPV117, SPPV122 and SPPV141 were also shown to induce formation of antibodies with virus-neutralizing activity. The results of the research will help to develop a new-generation high-performance means for specific sheep pox prophylaxis that is one of key moments in animal health protection. The research was conducted under the International project ISTC # K-1704 “Development of methods to construct recombinant prophylactic means for sheep pox with use of transgenic plants” and under the Grant Project RK MES G.2015/0115RK01983 "Recombinant vaccine for sheep pox prophylaxis".

Keywords: prophylactic preparation, recombinant protein, sheep pox virus, subunit vaccine

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Authors: Vera Lúcia Raposo

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The right to health care – usually known as the right to health – is recognized in many national laws and Constitutions, as well as in international human rights documents. The kind of health care that citizens are entitled to receive, especially in the framework of the National Health Service, is usually identified with conventional medicine. However, since ancient times that a different form of medicine – alternative, traditional or nonconventional medicine – exists. In recent times it is attracting increasing interest, as it is demonstrated by the use of its specific knowledge either by pharmaceutical companies either by modern health technologies. Alternative medicine refers to a holistic approach to body and mind using herbal products, animal parts and minerals instead of technology and pharmaceutical drugs. These notes contributed to a sense of distrust towards it, accusing alternative medicine of being based on superstition and ignorance. However, and without denying that some particular practices lack indeed any kind of evidence or scientific grounds, the fact is that a substantial part of alternative medicine can actually produce satisfactory results. The paper will not advocate the substitution of conventional medicine by alternative medicine, but the complementation between the two and their specific knowledge. In terms of the right to health, as a fundamental right and a human right, this thesis leads to the implementation of a wider range of therapeutic choices for patients, who should be entitled to receive different forms of health care that complement one another, both in public and private health facilities. This scenario would demand a proper regulation for alternative medicine, which nowadays does not exist in most countries, but it is essential to protect patients and public health in general and to reinforce confidence in alternative medicine.

Keywords: alternative medicine, conventional medicine, patient’s rights, right to health

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Authors: Bercem Kiran-Yildirim, Volker Gaukel

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Ice recrystallization (IR) which occurs especially during frozen storage is an undesired process due to the possible influence on the quality of products. As a result of recrystallization, the total volume of ice remains constant, but the size, number, and shape of ice crystals change. For instance, as indicated in the literature, the size of ice crystals in ice cream increases due to recrystallization. This results in texture deterioration. Therefore, the inhibition of ice recrystallization is of great importance, not only for food industry but also for several other areas where sensitive products are stored frozen, like pharmaceutical products or organs and blood in medicine. Ice-binding proteins (IBPs) have the unique ability to inhibit ice growth and in consequence inhibit recrystallization. This effect is based on their ice binding affinity. In the presence of IBP in a solution, ice crystal growth is inhibited during temperature decrease until a certain temperature is reached. The melting during temperature increase is not influenced. The gap between melting and freezing points is known as thermal hysteresis (TH). In literature, the TH activity is usually investigated under laboratory conditions in IBP buffer solutions. In product applications (e.g., food) there are many other solutes present which may influence the TH activity. In this study, a subset of IBPs, so-called antifreeze proteins (AFPs), is used for the investigation of the influence of sucrose solution concentration on the TH activity. For the investigation, a polarization microscope (Nikon Eclipse LV100ND) equipped with a digital camera (Nikon DS-Ri1) and a cold stage (Linkam LTS420) was used. In a first step, the equipment was established and validated concerning the accuracy of TH measurements based on literature data.

Keywords: ice binding proteins, ice crystals, sucrose solution, thermal hysteresis

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Authors: Samira Khalaji, , Fenneke Klein Jan, Kay-E. Gottschalk, Eugenia Makrantonaki, Karin Scharffetter-Kochanek

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Biological aging is a multi-dimensional process that takes place over a whole range of scales from the nanoscopic alterations within individual cells, over transformations in tissues and organs and to changes of the whole organism. On the single cell level, aging involves mutation of genes, differences in gene expression levels as well as altered posttranslational modifications of proteins. A variety of proteins is affected, including proteins of the cell cytoskeleton and migration machinery. Previous work quantified the expression of cytoskeleton proteins on the gene and protein levels in senescent and young fibroblasts. Their results show that senescent skin fibroblasts have an upregulated expression of the intermediate filament (IF) protein vimentin in contrast to actin and tubulin, which are downregulated. IFs play an important role in providing mechanical stability of cells. However, the mechanical properties of IFs depending on cellular senescence or age of the donor has not been studied so far. Hence, we employed passive microrheology on primary human dermal fibroblasts from female donors with age of 28 years (young) and 86 years (old) as model of in vivo aging and human normal dermal fibroblast from 11-year old male with CPD 17-35 (young) and CPD 58-59 (senescence) as a model of in vitro replicative senescence. In contrast to the expectations, our primary results show no significant differences in the viscoelastic properties of fibroblasts depending on age of the donor or cellular replicative senescence.

Keywords: aging, cytoskeleton, fibroblast, mechanical properties

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Authors: Tamer M. El-Messery

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By-products from sunflower oil extraction, such as sunflower cakes, are rich sources of proteins with desirable functional properties for the food industry. However, challenges such as sensory drawbacks and the presence of phenolic compounds have hindered their widespread use. In this study, sunflower protein concentrates were obtained from sunflower cakes using different ant-greening solvents (ascorbic acid (ASC) and N-acetylcysteine (NAC)), and their functional properties were evaluated. The color of extracted proteins ranged from dark green to yellow, where the using of ASC and NAC agents enhanced the color. The protein concentrates exhibited high solubility (>70%) and antioxidant activity, with hydrophobicity influencing emulsifying activity. Emulsions prepared with these proteins showed stability and microencapsulation efficiency. Incorporation of protein concentrates into low-fat whipping cream formulations increased overrun and affected color characteristics. Rheological studies demonstrated pseudoplastic behavior in whipped cream, influenced by shear rates and protein content. Overall, sunflower protein isolates showed promising functional properties, indicating their potential as valuable ingredients in food formulations.

Keywords: functional properties, sunflower protein concentrates, antioxidant capacity, ant-greening agents, low-fat whipping cream

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Authors: Maryam Ghamsari, Mitchell Nye-Wood, Kelvin Wang, Angela Juhasz, Michelle Colgrave, Don Otter, Jun Lu, Nazimah Hamid, Thao T. Le

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Bee venom, a complex mixture of peptides, proteins, enzymes, and other bioactive compounds, has been widely studied for its therapeutic application. This study investigated the proteins present in New Zealand (NZ) honeybee venom (BV) using bottom-up proteomics. Two sample digestion techniques, in-solution digestion and filter-aided sample preparation (FASP), were employed to obtain the optimal method for protein digestion. Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH–MS) analysis was conducted to quantify the protein compositions of NZ BV and investigate variations in collection years. Our results revealed high protein content (158.12 µg/mL), with the FASP method yielding a larger number of identified proteins (125) than in-solution digestion (95). SWATH–MS indicated melittin and phospholipase A2 as the most abundant proteins. Significant variations in protein compositions across samples from different years (2018, 2019, 2021) were observed, with implications for venom's bioactivity. In vitro testing demonstrated immunomodulatory and antioxidant activities, with a viable range for cell growth established at 1.5-5 µg/mL. The study underscores the value of proteomic tools in characterizing bioactive compounds in bee venom, paving the way for deeper exploration into their therapeutic potentials. Further research is needed to fractionate the venom and elucidate the mechanisms of action for the identified bioactive components.

Keywords: honeybee venom, proteomics, bioactivity, fractionation, swath-ms, melittin, phospholipase a2, new zealand, immunomodulatory, antioxidant

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Authors: Syeda Fahria Hoque Mimmi

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Many new and promising treatments for reducing or diminishing the adverse effects of microorganisms are being discovered day by day. On the other hand, the dairy industry is accelerating the economic wheel of Bangladesh. Considering all these facts, new thoughts were developed to isolate milk proteins by the present experiment for opening up a new era of developing natural antibiotics from milk. Lactoferrin, an iron-binding glycoprotein with multifunctional properties, is crucial to strengthening the immune system and also useful for commercial applications. The protein’s iron-binding capacity makes it undoubtedly advantageous to immune system modulation and different bacterial strains. For fulfilling the purpose, 4 of raw and 17 of commercially available milk samples were collected from different farms and stores in Bangladesh (Dhaka, Chittagong, and Cox’s Bazar). Protein quantification by nanodrop technology has confirmed that raw milk samples have better quantities of protein than the commercial ones. All the samples were tested for their antimicrobial activity against 18 pathogens, where raw milk samples showed a higher percentage of antibacterial activity. In addition to this, SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis) was performed to identify lactoferrin in the milk samples. Lactoferrin was detected in 9 samples from which 4 were raw milk samples. Interestingly, Streptococcus pyogenes, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, Vibrio cholera, Staphylococcus aureus, and enterotoxigenic E. coli significantly displayed sensitivity against lactoferrin collected from raw milk. Only Bacillus cereus, Pseudomonas aeruginosa, Streptococcus pneumonia, Enterococcus faecalis, and ETEC (Enterotoxigenic Escherichia coli) were susceptible to lactoferrin obtained from a commercial one. This study suggested that lactoferrin might be used as the potential alternative of antibiotics for many diseases and also can be used to reduce microbial deterioration in the food and feed industry.

Keywords: alternative of antibiotics, commercially available milk, lactoferrin, nanodrop technology, pathogens, raw milk

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Authors: Ahmed M. Hjazi, Bader M. Hjazi

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Protein 4.1 is a crucial component of complex interactions between the cytoskeleton and other junctional complex proteins. When the gene encoding this protein is altered, resulting in reduced expression, or when the protein is absent, the red cell undergoes a significant structural change. This research aims to achieve a deeper comprehension of the biochemical effects of red cell protein deficiency. A Tandem Mass Spectrometry Analysis (TMT-MS/MS) of patient cells lacking protein 4.1 compared to three healthy controls was achieved by the Proteomics Institute of the University of Bristol. The SDS-PAGE and Western blotting were utilized on the original patient sample and controls to partially confirm TMT MS/MS data analysis of the protein-4.1-deficient cells. Compared to healthy controls, protein levels in samples lacking protein 4.1 had a significantly higher concentration of proteins that probably originated from reticulocytes. This could occur if the patient has an elevated reticulocyte count. The increase in chaperone and reticulocyte-associated proteins was most notable in this study. This may result from elevated quantities of reticulocytes in patients with hereditary elliptocytosis.

Keywords: hereditary elliptocytosis, protein 4.1, red cells, tandem mass spectrometry data.

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Authors: Ahreum Choi, Otgontuya Tsogbadrakh, Kwang-Hwan Jung

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Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied. These retinal-binding proteins have divided into two types. The type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. For type I rhodopsin, there are two main functions that are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is one of the microbial rhodopsin with main function as photo-sensory transduction. Although ASR is expressed fairly well in Escherichia coli, the expression level is relatively less compare to Proteorhodopsin. In this study, full length of ASR was used to test for the expression influence by codon usage in E. coli. Eight amino acids of codon at N-terminal part of ASR were changed randomly with designed primers, which allow 8,192 nucleotide different cases. The codon changes were screened for the preferable codons of each residue, which have given higher expression yield. Among those 57 selected mutations, there are 24 color-enhanced E. coli colonies that contain ASR proteins, and it showed better expression level than the wild type ASR codon usage. This strongly suggests that high codon usage of only partial N-terminal of protein can increase the expression level of whole protein.

Keywords: 7-transmembrane, all-trans retinal, rhodopsin, codon-usage, protein expression

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Authors: Alina Goldberg Cavalleri, Sara Franco Ortega, Nawaporn Onkokesung, Richard Dale, Melissa Brazier-Hicks, Robert Edwards

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Non-target site based resistance (NTSR) to herbicides in weeds is a polygenic trait associated with the upregulation of proteins involved in xenobiotic detoxification and translocation we have termed the xenome. Among the xenome proteins, ABC transporters play a key role in enhancing herbicide metabolism by effluxing conjugated xenobiotics from the cytoplasm into the vacuole. The importance of ABC transporters is emphasized by the fact that they often contribute to multidrug resistance in human cells and antibiotic resistance in bacteria. They also play a key role in insecticide resistance in major vectors of human diseases and crop pests. By surveying available databases, transcripts encoding ABCs have been identified as being enhanced in populations exhibiting NTSR in several weed species. Based on a transcriptomics data in black grass (Alopecurus myosuroides, Am), we have identified three proteins from the ABC-C subfamily that are upregulated in NTSR populations. ABC-C transporters are poorly characterized proteins in plants, but in Arabidopsis localize to the vacuolar membrane and have functional roles in transporting glutathionylated (GSH)-xenobiotic conjugates. We found that the up-regulation of AmABCs strongly correlates with the up-regulation of a glutathione transferase termed AmGSTU2, which can conjugate GSH to herbicides. The expression profile of the ABC transcripts was profiled in populations of black grass showing different degree of resistance to herbicides. This, together with a phylogenetic analysis, revealed that AmABCs cluster in different groups which might indicate different substrate and roles in the herbicide resistance phenotype in the different populations

Keywords: black grass, herbicide, resistance, transporters

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Authors: Carolina V. Viegas, Monique Gonçalves, Gisel Chenard Diaz, Yordanka Reyes Cruz, Donato Alexandre Gomes Aranda

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To develop the massive cultivation of microalgae, it is necessary to isolate and characterize the species, improving genetic tools in search of specific characteristics. Therefore, the detection, identification and quantification of the compounds that compose the Scenedesmus sp. were prerequisites to verify the potential of these microalgae. The main objective of this work was to carry out the characterization of Scenedesmus sp. as to the content of ash, carbohydrates, proteins and lipids as well as the determination of the composition of their lipid classes and main fatty acids. The biomass of Scenedesmus sp, showed 15,29 ± 0,23 % of ash and CaO (36,17 %) was the main component of this fraction, The total protein and carbohydrate content of the biomass was 40,74 ± 1,01 % and 23,37 ± 0,95 %, respectively, proving to be a potential source of proteins as well as carbohydrates for the production of ethanol via fermentation, The lipid contents extracted via Bligh & Dyer and in situ saponification were 8,18 ± 0,13 % and 4,11 ± 0,11 %, respectively. In the lipid extracts obtained via Bligh & Dyer, approximately 50 % of the composition of this fraction consists of fatty compounds, while the other half is composed of an unsaponifiable fraction composed mainly of chlorophylls, phytosterols and carotenes. From the lowest yield, it was possible to obtain a selectivity of 92,14 % for fatty components (fatty acids and fatty esters) confirmed through the infrared spectroscopy technique. The presence of polyunsaturated acids (~45 %) in the lipid extracts indicated the potential of this fraction as a source of nutraceuticals. The results indicate that the biomass of Scenedesmus sp, can become a promising potential source for obtaining polyunsaturated fatty acids, carotenoids and proteins as well as the simultaneous obtainment of different compounds of high commercial value.

Keywords: microalgae, Desmodesmus, lipid classes, fatty acid profile, proteins, carbohydrates

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Authors: Lavinia Ruta, Ileana Farcasanu

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The yeast surface display (YSD) technique enables the expression of proteins on yeast cell surfaces, facilitating the identification and isolation of proteins with targeted binding properties, such as nanobodies. Nanobodies, derived from camelid species, are single-domain antibody fragments renowned for their high affinity and specificity towards target proteins, making them valuable in research and potentially in therapeutics. Their advantages include a compact size (~15 kDa), robust stability, and the ability to target challenging epitopes. The project endeavors to establish and validate a platform for producing Green Fluorescent Protein (GFP) and mCherry nanobodies using the yeast surface display method. mCherry, a prevalent red fluorescent protein sourced from coral species, is commonly utilized as a genetic marker in biological studies due to its vibrant red fluorescence. The GFP-nanobody, a single variable domain of heavy-chain antibodies (VHH), exhibits specific binding to GFP, offering a potent means for isolating and engineering fluorescent protein fusions across various biological research domains. Both GFP and mCherry nanobodies find specific utility in cellular imaging and protein analysis applications.

Keywords: YSD, nanobodies, GFP, Saccharomyces cerevisiae

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Authors: Daniel Zarzo Montes, Manuel Zarzo Castelló

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Proteins play a fundamental role in biology, but their structure is complex, and it is a challenge for teachers to conceptually explain the differences between their primary, secondary, tertiary, and quaternary structures. On the other hand, there are currently many computer programs to visualize the 3D structure of proteins, but they require advanced training and knowledge. Moreover, it becomes difficult to visualize the sequence of amino acids in these models, and how the protein conformation is reached. Given this drawback, a simple and instructive procedure is proposed in order to teach the protein structure to undergraduate and graduate students. For this purpose, insulin has been chosen because it is a protein that consists of 51 amino acids, a relatively small number. The methodology has consisted of the use of plastic atom models, which are frequently used in organic chemistry and biochemistry to explain the chirality of biomolecules. For didactic purposes, when the aim is to teach the biochemical foundations of proteins, a manipulative system seems convenient, starting from the chemical structure of amino acids. It has the advantage that the bonds between amino acids can be conveniently rotated, following the pattern marked by the 3D models. First, the 51 amino acids were modeled, and then they were linked according to the sequence of this protein. Next, the three disulfide bonds that characterize the stability of insulin have been established, and then the alpha-helix structure has been formed. In order to reach the tertiary 3D conformation of this protein, different interactive models available on the Internet have been visualized. In conclusion, the proposed methodology seems very suitable for biology and biochemistry students because they can learn the fundamentals of protein modeling by means of a manipulative procedure as a basis for understanding the functionality of proteins. This methodology would be conveniently useful for a biology or biochemistry laboratory practice, either at the pre-graduate or university level.

Keywords: protein structure, 3D model, insulin, biomolecule

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Authors: V. C. Santos-Ebinuma, J. F. B. Pereira, M. F. S. Teixeira, A. Pessoa Jr., P. Jauregi

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There is worldwide interest in process development for colorants production from natural sources. Microorganisms provide an alternative source of natural colorants which can be produced by cultivation technology and extracted from fermented broth. The aim of the present work was to study the recovery of red colorants from fermented broth of Penicillium purpurogenum DPUA 1275 using the technique of Colloidal Gas Aphrons (CGA); CGA are surfactant-stabilized microbubbles generated by intense stirring of a surfactant solution. CGA were generated by the cationic, hexadecyl trimethyl ammonium bromide (CTAB) surfactant. Firstly, experiments were carried out at different surfactant/fermented broth volumetric ratios (VCGA/VFB, VRATIO) varying between 3 and 18 at pH 6.9. Secondly, the experiments were carried out at VRATIO of 6 and 12 in different pH, namely, 6.9, 8.0, 9.0 and 10.0. The first results of recovery showed that an increase in the VRATIO from 3 to 6 and 12 promoted an increase as recovery as partition coefficient. However, at VRATIO of 18 the lowest partition coefficient was obtained. The best results were achieved at VRATIO of 6 and 12, namely recovery, Re, around 60% and partition coefficient, K, of 2.5 and 3.0 to 6 and 12 VRATIO, respectively. The second set of experiments showed that the pH 9.0 promoted the best results at VRATIO of 12 as follow: Re=70%, K=5.39, proteins and sugar selectivity (SePROT, 3.75 and SeSUGAR, 7.20, respectively). These results indicate that with CTAB the recovery is mainly driven by electrostatic interactions. In conclusion, the results above show that CGA employing a cationic surfactant is a promissory technique and it can be used as the first step of purification to recovery red colorants from fermented broth.

Keywords: liquid-liquid extraction, colloidal gas aphrons, recovery, natural colorants

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Authors: Abdelbary Prince, Said Moussa, Hyungkyu Kim, Eman Gouda, Jin Han

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We compared the dynamic alterations of mitochondrial proteome of control, ischemia-reperfusion (IR) and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 29 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For two of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included succinate dehydrogenase complex, Acyl-CoA dehydrogenase, carnitine acetyltransferase, dihydrolipoamide dehydrogenase, Atpase, ATP synthase, dihydrolipoamide succinyltransferase, ubiquinol-cytochrome c reductase, translation elongation factor, acyl-CoA dehydrogenase, actin alpha, succinyl-CoA Ligase, dihydrolipoamide S-succinyltransferase, citrate synthase, acetyl-Coenzyme A dehydrogenase, creatine kinase, isocitrate dehydrogenase, pyruvate dehydrogenase, prohibitin, NADH dehydrogenase (ubiquinone) Fe-S protein, enoyl Coenzyme A hydratase, superoxide dismutase [Mn], and 24-kDa subunit of complex I. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain, antioxidant enzyme system, and energy metabolism. The results provide clues as to the cardioprotective mechanism of ischemic preconditioning at the protein level and may serve as potential biomarkers for detection of ischemia-induced cardiac injury.

Keywords: ischemic preconditioning, mitochondria, proteome, cardioprotection

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Authors: Mario Hanna Louis Hanna

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The use of CAD (pc Aided layout) generation is ubiquitous inside the structure, engineering and construction (AEC) industry. This has led to its inclusion in the curriculum of structure faculties in Nigeria as an important part of the training module. This newsletter examines the moral troubles involved in implementing CAD (pc Aided layout) content into the architectural training curriculum. Using current literature, this study begins with the advantages of integrating CAD into architectural education and the responsibilities of various stakeholders in the implementation process. It also examines issues related to the terrible use of records generation and the perceived bad effect of CAD use on design creativity. The use of a survey technique, information from the architecture department of Chukwuemeka Odumegwu Ojukwu Uli college changed into accumulated to serve as a case observe on how the problems raised have been being addressed. The object draws conclusions on what guarantees a hit moral implementation. Tens of millions of human beings around the sector suffer from hepatitis C, one of the international's deadliest sicknesses. Interferon (IFN) is a remedy alternative for patients with hepatitis C, but these treatments have their aspect outcomes. Our research targeted growing an oral small molecule drug that goals hepatitis C virus (HCV) proteins and has fewer facet effects. Our contemporary study targets to broaden a drug primarily based on a small molecule antiviral drug precise for the hepatitis C virus (HCV). Drug improvement and the use of laboratory experiments isn't always best high-priced, however also time-eating to behavior those experiments. instead, on this in silicon have a look at, we used computational strategies to recommend a particular antiviral drug for the protein domain names of discovered in the hepatitis C virus. This examines used homology modeling and abs initio modeling to generate the 3-D shape of the proteins, then figuring out pockets within the proteins. Proper lagans for pocket pills were advanced the usage of the de novo drug design method. Pocket geometry is taken into consideration while designing ligands. A few of the various lagans generated, a different for each of the HCV protein domains has been proposed.

Keywords: drug design, anti-viral drug, in-silicon drug design, Hepatitis C virus (HCV) CAD (Computer Aided Design), CAD education, education improvement, small-size contractor automatic pharmacy, PLC, control system, management system, communication.

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Authors: Kim Byeong-Gon, Lee Pureun-Haneul, Hong Jisu, Jang An-Soo

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Background: Diesel exhaust particles (DEPs) lead to trigger airway hyperresponsiveness (AHR) and airway dysfunction or inflammation in respiratory systems. Whether tight junction protein changes can contribute to development or exacerbations of airway diseases remain to be clarified. Objective: The aim of this study was to observe the effect of DEP on tight junction proteins in one airway both nose and lung in a mouse model. Methods: Mice were treated with saline (Sham) and exposed to 100 μg/m³ DEPs 1 hour a day for 5 days a week for 4 weeks and 8 weeks in a closed-system chamber attached to a ultrasonic nebulizer. Airway hyperresponsiveness (AHR) was measured and bronchoalveolar lavage (BAL) fluid, nasal lavage (NAL) fluid, lung and nasal tissue was collected. The effects of DEP on tight junction proteins were estimated using western blot, immunohistochemical in lung and nasal tissue. Results: Airway hyperresponsiveness and number of inflammatory cells were higher in DEP exposure group than in control group, and were higher in 4 and 8 weeks model than in control group. The expression of tight junction proteins CLND4, -5, and -17 in both lung and nasal tissue were significantly increased in DEP exposure group than in the control group. Conclusion: These results suggesting that CLDN4, -5 and -17 may be involved in the airway both nose and lung, suggesting that air pollutants cause to disruption of epithelial and endothelial cell barriers. Acknowledgment: This research was supported by Korea Ministry of Environment (MOE) as 'The Environmental Health Action Program' (2016001360009) and Soonchunhyang University Research Fund.

Keywords: diesel exhaust particles, air pollutant, tight junction, Claudin, Airway inflammation

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Authors: Mohd Adil, Rosina Khan, Danishuddin, Asad U. Khan

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The aim of this study was to evaluate the influence of the crude and active solvent fraction of Trachyspermum ammi on S. mutans cariogenicity, effect on expression of genes involved in biofilm formation and caries development in rats. GC–MS was carried out to identify the major components present in the crude and the active fraction of T. ammi. The crude extract and the solvent fraction exhibiting least MIC were selected for further experiments. Scanning electron microscopy was carried out to observe the effect of the extracts on S. mutans biofilm. Comparative gene expression analysis was carried out for nine selected genes. 2-Isopropyl-5-methyl-phenol was found as major compound in crude and the active fraction. Binding site of this compound within the proteins involved in biofilm formation was mapped with the help of docking studies. Real-time RT-PCR analyses revealed significant suppression of the genes involved in biofilm formation. All the test groups showed reduction in caries (smooth surface as well as sulcal surface caries) in rats. Moreover, it also provides new insight to understand the mechanism influencing biofilm formation in S. mutans. Furthermore, the data suggest the putative cariostatic properties of T. Ammi and hence can be used as an alternative medicine to prevent caries infection.

Keywords: bio-film, Streptococcus mutans, dental caries, bio-informatic

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Authors: Valentin Nicorescu, Nicoleta C. Predescu, Camelia Papuc, Iuliana Gajaila, Carmen D. Petcu

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The oxidation of the fresh muscle oxymyoglobin (bright red colour) to metmyoglobin (brown colour) leads to discoloration of red meats. After slaughter, enzymatic systems involved in metmyoglobin reduction are continually depleted as time post-mortem progresses, thus the meat colour is affected. Phenolic compounds are able to scavenge reactive species involved in oxymyoglobin oxidation and to reduce metmyoglobin to oxymyoglobin. The aim of this study was to investigate the effect of polyphenols extracted from hawthorn fruits on the stability of oxymyoglobin and myofibrillar proteins in ground pork subject to refrigeration for 6 days. Hawthorn polyphenols (HP) were added in ground pork in 100, 200 and 300 ppm concentrations. Oxymyoglobin and metmyoglobin were evaluated spectrophotometrically at every 2 days and electrophoretic pattern of myofibrillar proteins was investigated at days 0 and 6 by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). For all meat samples, oxymyoglobin concentration significantly decreased during the first 4 days of refrigeration. After 6 days, the significant decrease of oxymyoglobin concentration continued only in the negative control samples. In samples treated with HP and butylated hydroxylanisole (BHA - positive control), oxymyoglobin concentration increased after 6 days of refrigeration, the highest levels complying with the following order: 100 ppm HP > 200 ppm HP > 300 ppm HP > 100 ppm BHA. The increase in metmyoglobin was coincidental with the decrease in oxymyoglobin; metmyoglobin concentration progressively increased during the first 4 days of refrigeration in all meat samples. After 6 days, in meat samples treated with HP and BHA, lower metmyoglobin concentrations were found (compared to day 4), respecting the following order: 100 ppm HP < 200 ppm HP < 300 ppm HP < 100 ppm BHA. These results showed that hawthorn polyphenols and BHA reduced metmyoglobin (MbFe3+) to oxymyoglobin (MbFe2+), and the strongest reducing character was recorded for 100 ppm HP. After 6 days of refrigeration, electrophoretic pattern of myofibrillar proteins showed minor changes compared to day 0, indicating that HP prevent protein degradation as well as synthetic antioxidant BHA. Also, HP did not induce cross-links in the myofibrillar proteins, to form protein aggregates, and no risk of reducing their ability to retain water was identified. The pattern of oxymyoglobin and metmyoglobin concentrations determined in this study showed that hawthorn polyphenols are able to reduce metmyoglobin to oxymyoglobin and to delay oxymyoglobin oxidation, especially when they are added to ground meat in concentration of 100 ppm. This work was carried out through Partnerships in priority areas Program – PN II, implemented with the support of MEN – UEFISCDI (Romania), project nr. 149/2014.

Keywords: Hawthorn polyphenols, metmyoglobin, oxymyoglobin, proteins stability

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Authors: M. Grifoni, G. Petruzzelli, M. Barbafieri, I. Rosellini, B. Pezzarossa, F. Pedron

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Evapotranspiration (ET) covers are an alternative cover system that utilizes water balance approach to maximize the ET process to reduce the contaminants leaching through the soil profile. Microcosm tests allow to identify in a short time the most suitable plant species to be used as alternative covers, their survival capacity, and simultaneously the transpiration and evaporation rate of the cover in a specific contaminated soil. This work shows the soil characterization and ET results of microcosm tests carried out on two contaminated soils by using Triticum durum and Helianthus annuus species. The data indicated that transpiration was higher than evaporation, supporting the use of plants as alternative cover at this contaminated site.

Keywords: contaminated sites, evapotranspiration cover, evapotranspiration, microcosm experiments

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Authors: S. Khosravi, X. Chelius, A. Unger, D. Rieger, J. Frickel, T. Sachsenheimer, C. Luechtenborg, R. Schieweck, B. Bruegger, B. Westermann, T. Klecker, W. Neupert, M. E. Harner

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The use of Saccharomyces cerevisiae as a model organism to study eukaryotic cell functions has been used successfully for decades. Like virtually all eukaryotic cells, they contain mitochondria as essential organelles performing various functions, including participation in lipid metabolism. They are separated from the cytosol by a double membrane system consisting of the mitochondrial inner membrane (MIM) and the mitochondrial outer membrane (MOM). This physical separation of the mitochondria requires an exchange of metabolites, proteins, and lipids. Proteinaceous contact sites are thought to be important for this communication. Recently, it was found that Cqd1, in cooperation with Cqd2, controls the distribution of Coenzyme Q within the cell. In this study, a contact site is described, formed by the MOM protein complex Por1-Om14 and the UbiB protein kinase-like MIM protein Cqd1. The present results suggest the additional involvement of Cqd1 in the homeostasis of phospholipids. Moreover, we show that overexpression of the UbiB family proteins also causes tethering of the mitochondria to the endoplasmatic reticulum. Due to the conservation of the subunits of this contact site to higher eukaryotes, its identification in S. cerevisiae might provide promising avenues for further research in other organisms.

Keywords: contact sites, mitochondrial architecture, mitochondrial proteins, yeast mitochondria

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Authors: S. Junaid S. Qazi, Denice C. Bay, Raymond Chew, Raymond J. Turner

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EmrE, a member of the small multidrug resistance protein family in bacteria is considered to be the archetypical member of its family. It confers host resistance to a wide variety of quaternary cation compounds (QCCs) driven by proton motive force. Generally, purification yield is a challenge in all membrane proteins because of the difficulties in their expression, isolation and solubilization. EmrE is extremely hydrophobic which make the purification yield challenging. We have purified EmrE protein using two different approaches: organic solvent membrane extraction and hexahistidine (his6) tagged Ni-affinity chromatographic methods. We have characterized changes present between ligand affinity of untagged and his6-tagged EmrE proteins in similar membrane mimetic environments using biophysical experimental techniques. Purified proteins were solubilized in a buffer containing n-dodecyl-β-D-maltopyranoside (DDM) and the conformations in the proteins were explored in the presence of four QCCs, methyl viologen (MV), ethidium bromide (EB), cetylpyridinium chloride (CTP) and tetraphenyl phosphonium (TPP). SDS-Tricine PAGE and dynamic light scattering (DLS) analysis revealed that the addition of QCCs did not induce higher multimeric forms of either proteins at all QCC:EmrE molar ratios examined under the solubilization conditions applied. QCC binding curves obtained from the Trp fluorescence quenching spectra, gave the values of dissociation constant (Kd) and maximum specific one-site binding (Bmax). Lower Bmax values to QCCs for his6-tagged EmrE shows that the binding sites remained unoccupied. This lower saturation suggests that the his6-tagged versions provide a conformation that prevents saturated binding. Our data demonstrate that tagging an integral membrane protein can significantly influence the protein.

Keywords: small multidrug resistance (SMR) protein, EmrE, integral membrane protein folding, quaternary ammonium compounds (QAC), quaternary cation compounds (QCC), nickel affinity chromatography, hexahistidine (His6) tag

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Authors: Mohammad Aladwan

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Alzheimer’s disease (AD) is a widespread dementing illness with a complex and poorly understood etiology. An important role in improving our understanding of the AD process is the modeling of disease-associated changes in tau protein phosphorylation, a protein known to mediate events essential to the onset and progression of AD. A main feature of AD is the abnormal phosphorylation of tau protein and the presence of neurofibrillary tangles. In order to evaluate the respective roles of the microtubule-binding region (MTBR) and alternatively spliced exons in the N-terminal projection domains in AD, we have constructed SHSY-5Y cell lines that stably overexpress four different species of tau protein (4R2N, 4R0N, N(E-2), N(E+2)). Since the toxicity and spreading of tau lesions in AD depends on the interactions of tau with other proteins, we have performed a proteomic analysis of exosome-fraction interactomes for cell lysates and media samples that were isolated from SHSY-5Y cell lines. Functional analysis of tau interactomes based on gene ontology (GO) terms was performed using the String 10.5 database program. The highest number of exosomes proteomes and tau associated proteins were found with 4R2N isoform (2771 and 159) in cell lysate and they have a high strength of connectivity (78%) between proteins, while N(E-2) isoform in the media proteomes has the highest number of proteins and tau associated protein (1829 and 205). Moreover, known AD markers were significantly enriched in secreted interactomes relative to lysate interactomes in the SHSY-5Y cells of tau isoforms lacking exons 2 and 3 in the N-terminal. The lack of exon 2 (E-2) from tau protein can be mediated by tau secretion and spreading to different cells. Enriched functions in the secreted E-2 interactome include signaling and developmental pathways that have been linked to a) tau misprocessing and lesion development and b) tau secretion and which, therefore, could play novel roles in AD pathogenesis.

Keywords: Alzheimer's disease, dementia, tau protein, neurodegenration disease

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