Search results for: milk proteins

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

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

Keywords: plasma, hemoglobin, myoglobin, solvent access

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

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

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

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Authors: 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

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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

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Authors: Ava Faridi, Pouya Faridi, Aleksandr Kakinen, Ibrahim Javed, Thomas P. Davis, Pu Chun Ke

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Human islet amyloid polypeptide (IAPP) is a hormone associated with glycemic control and type 2 diabetes. Biophysically, the chirality of IAPP fibrils has been little explored with respect to the aggregation and toxicity of the peptide. Biochemically, it remains unclear as for how protein expression in pancreatic beta cells may be altered by cell exposure to the peptide, and how such changes may be mitigated by nanoparticle inhibitors for IAPP aggregation. In this study, we first demonstrated the elimination of the IAPP nucleation phase and shortening of its elongation phase by silica nanoribbons. This accelerated IAPP fibrillization translated to reduced toxicity, especially for the right-handed silica nanoribbons, as revealed by cell viability, helium ion microscopy, as well as zebrafish embryo survival, developmental and behavioral assays. We then examined the proteomes of βTC6 pancreatic beta cells exposed to the three main aggregation states of monomeric, oligomeric and amyloid fibrillar IAPP, and compared that with cellular protein expression modulated by graphene quantum dots (GQDs). A total of 29 proteins were significantly regulated by different forms of IAPP, and the majority of these proteins were nucleotide-binding proteins. A regulatory capacity of GQDs against aberrant protein expression was confirmed. These studies have demonstrated the great potential of employing nanomaterials targeting the mesoscopic enantioselectivity and protein expression dysregulation in pancreatic beta cells.

Keywords: graphene quantum dots, IAPP, silica nanoribbons, protein expression, toxicity

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Authors: Kieren Luellman, Makenzi Rockwell, Eduardo Callegari, Nichole Haag, Chun Wu

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Multiple sclerosis (MS) is an autoimmune disorder that damages the myelin sheath of neurons in the central nervous system. The presence of Acinetobacter bacteria and anti-Acinetobacter antibodies in MS patients has led to the hypothesis that the bacteria may contribute to MS pathogenesis. In this study, the protein sequences of Acinetobacter baumannii were compared to five peptides from three mammalian myelin proteins, i.e., Proteolipid Protein (PLP): PLP 139-151, PLP 178-191, Myelin Basic Protein (MBP): MBP 84-104 and Myelin Oligodendrocyte Glycoprotein (MOG): MOG 35-55 and MOG 92-106 respectively, known to induce experimental autoimmune encephalomyelitis (EAE), a condition similar to MS. We found 11 hits (i.e., with five or more amino acid sequence similarity) in Acinetobacter baumannii, which are identical or similar to PLP139-151, 32 hits to PLP178-191, 35 to MBP 84-104, 41 hits to MOG 35-55 and 26 hits to MOG92-106. In addition, Western blotting was used to assess possible interaction between the bacterial proteins and human anti-MBP, anti-MOG, and anti-PLP antibodies produced in rabbits, corresponding to MBP 84-104, MOG 35-55, and PLP 139-151, respectively. We found that both human Polyclonal anti-MOG antibody and anti-PLP antibody recognized a protein or more proteins of the same molecular mass of around 25 kDa. in Acinetobacter baumannii. The results suggested that this/these protein(s) might potentially serve as antigen(s) to induce anti-MOG antibody and anti-PLP antibody production in mammalian B cells. The proteomic study identified 433 hits, among which the sequence of Acinetobacter baumannii protein 491 subunit A matches a previously published enzyme Acinetobacter 3-Oxoadipate CoA-Transferase, in which a fragment of its peptide was observed to recognize MS patient serum via ELISA method. Our findings might pave the road to understanding one of the pathogenesis mechanisms of MS.

Keywords: multiple sclerosis, pathogenesis, Acinetobacter baumannii, antibody recognition

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Authors: M. Dehestani, F. Kamali, M. Klantari Pour, L. Zeidabadi-Nejad

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Chemical bonding between polyethylene glycol (PEG) with pharmaceutical proteins called pegylation is one of the most effective methods of improving the pharmacological properties. The covalent attachment of polyethylene glycol (PEG) to proteins will increase their pharmacologic properties. For the formation of a combination of pegylated protein should first be activated PEG and connected to the protein. Interferons(IFNs) are a family of cytokines which show antiviral effects in front of the biological and are responsible for setting safety system. In this study, the nature and properties of the interaction between active positions of IFNs and polyethylene glycol have been investigated using molecular dynamics simulation. The main aspect of this theoretical work focuses on the achievement of valuable data on the reaction pathways of PEG-IFNs and the transition state energy. Our results provide a new perspective on the interactions, chemical properties and reaction pathways between IFNs and PEG.

Keywords: interaction, interferons, molecular dynamics simulation, polyethylene glycol

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Authors: Mayuva Youngsabanant, Chanikarn Srinark, Supanyika Sengsai, Soratorn Kerdkriangkrai, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The follicular fluid proteins of healthy medium size follicles (4-6 mm in diameters) and large size follicles (7-8 mm in diameter) of large white pig ovaries were collected by using sterile technique. They were used for testing the effect on primary in vitro cell culture growth of porcine oviductal epithelial cells (pOEC). Porcine oviductal epithelial cells of luteal phase was culture in M199 and added with 10% fetal calf serum 2.2 mg/mL, NaHCO₃, 0.25 mM pyruvate, 15 µg/mL and 50 µg/mL, gentamycin sulfate at high humidified atmosphere with 5% CO₂ in 95% air atmosphere at 37°C for 96 h before testing. The optimized concentration of pFF of two follicle sizes (at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins) in culture medium was observed for 24 h using MTT assay. Results were analyzed with a one-way ANOVA in SPSS statistic. Moreover, pOEC was also studied in morphological characteristic on long-term culture. The results of long-term study revealed that pOEC showed 70-80 percentage of healthy morphology on epithelial-like character and contained 30 percentage of an elongated shape (fibroblast-like morphology) at 4 weeks of culture time. MTT assay reviewed an increase in the percentage of viability of pOEC in 2 treated of follicular fluid groups. Two treatment concentration groups were higher than control group (p < 0.05) but not in positive control group. Interestingly, at 200 µg protein of 2 treated follicular fluid groups were reached the highest cell viability which is higher than a positive control and it is significantly different form control group (P < 0.05). These cells are developed and had fibroblast elongate shape which is longer than the cells in control group and positive control group. This report implies that pFF of medium follicle size at 200 µg proteins and large follicle size at 200 and 500 µg proteins could be optimized concentration for using as a supplement in culture medium to promote cell growth and development instead of growth hormone from fetal calf serum. It could be applied in cell biotechnology researches. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: in vitro, porcine follicular fluid protein (pFF), porcine oviductal epithelial cells (pOEC), MTT

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Authors: Bouchiha Hanene, Rouabhi Rachid, Bouchama Khaled, Djebar Berrebbah Houraya, Djebar Mohamed Reda

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Recently, some natural products such as essentials oils (EOs) have been used in the fields as alternative to synthetic compounds, to minimize the negative impacts to the environment. This fact has led to questions about the possible impact of EOs on ecosystems. Currently in toxicology, the use of alternative models can help to understand the mechanisms of toxic action, at different levels of organization of ecosystems. Algae, protozoa and bacteria form the base of the food chain and protozoan cells are used as bioindicators often of pollution in environment. Unicellular organisms offer the possibility of direct study of independent cells with specific characteristics of individual cells and whole organisms at the same time. This unicellular facilitates the study of physiological processes, and effects of pollutants at the cellular level, which makes it widely used to assess the toxic effects of various xenobiotics. This study aimed to verify the effects of EOs of one famous plant used tremendously in our folk medicine, namely Artemisia herba alba in causing acute toxicity (24 hours) and chronic (15 days) toxicity for model cellular (Paramecium sp). To this end, cellular’s of paramecium were exposed to various concentrations (Three doses were chosen) of EOs extracted from plant (Artemisia herba alba). In the first experiment, the cellular s cultures were exposed for 48 hours to different concentrations to determine the median lethal concentration (DL50). We followed the evolution of physiological parameters (growth), biochemical (total proteins, respiratory metabolism), as well as the variations of a bio marker the GSH. Our results highlighted a light inhibition of the growth of the protozoa as well as a disturbance of the contents of total proteins and a reduction in the reduced rate of glutathione. The polarographic study revealed a stimulation of the consumption of O2 and this at the treated cells.

Keywords: essential oils, protozoa, bio indicators, toxicity, Growth, bio marker, proteins, polarographic

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Authors: Qing Cao, Fei Wang, Yu-Qiang Wang, Li-Ya Huang, Tian-Tian Sang, Shu-Yan Chen

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Aims: TNF Receptor-Associated Factor 6 (TRAF6) acts as a multifunctional regulator of the Transforming Growth Factor (TGF)-β signaling pathway, and mediates Smad-independent JNK and p38 activation via TGF-β. This study was performed to test the hypothesis that TGF-β/TRAF6 is essential for angiotensin-II (Ang II)-induced differentiation of rat c-kit+ Cardiac Stem Cells (CSCs). Methods and Results: c-kit+ CSCs were isolated from neonatal Sprague Dawley (SD) rats, and their c-kit status was confirmed with immunofluorescence staining. A TGF-β type I receptor inhibitor (SB431542) or the small interfering RNA (siRNA)-mediated knockdown of TRAF6 were used to investigate the role of TRAF6 in TGF-β signaling. Rescue of TRAF6 siRNA transfected cells with a 3'UTR deleted siRNA insensitive construct was conducted to rule out the off target effects of the siRNA. TRAF6 dominant negative (TRAF6DN) vector was constructed and used to infect c-kit+ CSCs, and western blotting was used to assess the expression of TRAF6, JNK, p38, cardiac-specific proteins, and Wnt signaling proteins. Physical interactions between TRAF6 and TGFβ receptors were studied by coimmunoprecipitation. Cardiac differentiation was suppressed in the absence of TRAF6. Forced expression of TRAF6 enhanced the expression of TGF-β-activated kinase1 (TAK1), and inhibited Wnt signaling. Furthermore, TRAF6 increased the expression of cardiac-specific proteins (cTnT and Cx-43) but inhibited the expression of Wnt3a. Conclusions: Our data suggest that TRAF6 plays an important role in Ang II induced differentiation of c-kit+ CSCs via the non-canonical signaling pathway.

Keywords: cardiac stem cells, differentiation, TGF-β, TRAF6, ubiquitination, Wnt

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Authors: Harika Atmaca, Emir Bozkurt, Latife Merve Oktay, Selim Uzunoglu, Ruchan Uslu, Burçak Karaca

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Microarrays have been developed for highly parallel enzyme-linked immunosorbent assay (ELISA) applications. The most common protein arrays are produced by using multiple monoclonal antibodies, since they are robust molecules which can be easily handled and immobilized by standard procedures without loss of activity. Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate, Ecteinascidia turbinata, has been shown to have antitumor effects. Here, we used a novel proteomic approach to explore the mechanism of action of trabectedin in prostate cancer cell line PC-3 by apoptosis antibody microarray. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. Trabectedin induced cytotoxicity and apoptosis in prostate cancer cells in a time and concentration-dependent manner. The expression levels of the death receptor pathway molecules, TRAIL-R1/DR4, TRAIL R2/DR5, TNF R1/TNFRSF1A, FADD were significantly increased by 4.0-, 21.0-, 4.20- and 11.5-fold by trabectedin treatment in PC-3 cells. Moreover, mitochondrial pathway related pro-apoptotic proteins Bax, Bad, Cytochrome c, and Cleaved Caspase-3 expressions were induced by 2.68-, 2.07-, 2.8-, and 4.5-fold and the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-XL were reduced by 3.5- and 5.2-fold in PC-3 cells. Proteomic (antibody microarray) analysis suggests that the mechanism of action of trabectedin may be exerted via the induction of both intrinsic and extrinsic apoptotic pathways. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents.

Keywords: trabectedin, prostate cancer, omic protein expression profile, apoptosis

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Authors: Sahar Essa, Hussain A. Safar, Raj Raghupathy

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Human cytomegalovirus (CMV) continues to be a source of severe complications in immunologically immature and immunocompromised hosts. Effective CMV vaccines that help diminish CMV disease in transplant patients and avoid congenital infection are of great importance. Though the exact roles of defense mechanisms are unidentified, viral-specific antibodies and cytokine responses are known to be involved in controlling CMV infections. CMV envelope glycoprotein B (UL55/gB), matrix proteins (UL83/pp65, UL99/pp28, UL32/pp150), and assembly protein UL80a/pp38 are known to be targets of antiviral immune responses. We immunized mice intraperitoneally with these five CMV-related proteins (commercial) for their ability to induce specific antibody responses (in-house immunoassay) and cytokine production (commercial assay) in a mouse model. We observed a significant CMV-antigen-specific antibody response to pp38 and pp65 (E/C ˃2.0, p˂0.001). Mice immunized with pp38 had significantly higher concentrations of GM-CSF, IFN-α, IL-2 IL-4, IL-5, and IL-17A (p˂0.05). Mice immunized with pp65 showed significantly higher concentrations of GM-CSF, IFN-γ, IL-2 IL-4, IL-10, IL-12, IL-17A, and TNF-α. Th1 to Th2 cytokines ratios revealed a Th1 cytokine bias in mice immunized with pp38, pp65, pp150, and gB. We suggest that stimulation with multiple CMV-related proteins, which include pp38, pp65, and gB antigens, will allow both humoral and cellular immune responses to be efficiently activated, thus serving as appropriate CMV antigens for future vaccines.

Keywords: cytomegalovirus, UL99/pp28, UL80a/pp38, UL83/pp65, UL32/pp150, UL55/gB, CMV-antigen-specific antibody, CMV antigen-specific cytokine responses

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Authors: Andrés Lara Guillén, José M. Soriano Disla, Gemma Castejón Martínez, David Fernández-Gutiérrez

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The worldwide population is exponentially increasing, which causes a rising demand for food, energy and non-renewable resources. These demands must be attended to from a circular economy point of view. Under this approach, the obtention of strategic products from biowaste is crucial for the society to keep the current lifestyle reducing the environmental and social issues linked to the lineal economy. This is the main objective of the VALUEWASTE project. VALUEWASTE is about valorizing urban biowaste into proteins for food and feed and biofertilizers, closing the loop of this waste stream. In order to achieve this objective, the project validates three value chains, which begin with the anaerobic digestion of the biowaste. From the anaerobic digestion, three by-products are obtained: i) methane that is used by microorganisms, which will be transformed into microbial proteins; ii) digestate that is used by black soldier fly, producing insect proteins; and iii) a nutrient-rich effluent, which will be transformed into biofertilizers. VALUEWASTE is an innovative solution, which combines different technologies to valorize entirely the biowaste. However, it is also required to demonstrate that the solution is greener than other traditional technologies (baseline systems). On one hand, the proteins from microorganisms and insects will be compared with other reference protein production systems (gluten, whey and soybean). On the other hand, the biofertilizers will be compared to the production of mineral fertilizers (ammonium sulphate and synthetic struvite). Therefore, the aim of this study is to provide that biowaste valorization can reduce the environmental impacts linked to both traditional proteins manufacturing processes and mineral fertilizers, not only at a pilot-scale but also at an industrial one. In the present study, both baseline system and VALUEWASTE solution are evaluated through the Environmental Life Cycle Assessment (E-LCA). The E-LCA is based on the standards ISO 14040 and 14044. The Environmental Footprint methodology was the one used in this study to evaluate the environmental impacts. The results for the baseline cases show that the food proteins coming from whey have the highest environmental impact on ecosystems compared to the other proteins sources: 7.5 and 15.9 folds higher than soybean and gluten, respectively. Comparing feed soybean and gluten, soybean has an environmental impact on human health 195.1 folds higher. In the case of biofertilizers, synthetic struvite has higher impacts than ammonium sulfate: 15.3 (ecosystems) and 11.8 (human health) fold, respectively. The results shown in the present study will be used as a reference to demonstrate the better environmental performance of the bio-based products obtained through the VALUEWASTE solution. Other originalities that the E-LCA performed in the VALUEWASTE project provides are the diverse direct implications on investment and policies. On one hand, better environmental performance will serve to remove the barriers linked to these kinds of technologies, boosting the investment that is backed by the E-LCA. On the other hand, it will be a germ to design new policies fostering these types of solutions to achieve two of the key targets of the European Community: being self-sustainable and carbon neutral.

Keywords: anaerobic digestion, biofertilizers, circular economy, nutrients recovery

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Authors: A. K. Tursunova, O. V. Chebonenko, A. Zh. Amirkulova, A. O. Abaildayev, O. A. Sapko, Y. M. Dyo, A. Sh. Utarbaeva

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Fusarium solani and its variants cause root and stem rot of plants. Dry rot is the most common disease of potato tubers during storage. The causative agents of fusariosis in contact with plants behave as antagonists, growth stimulants or parasites. The diversity of host-parasite relationships is explained by the parasite’s ability to produce a wide spectrum of biologically active compounds including toxins, enzymes, oligosaccharides, antibiotic substances, enniatins and gibberellins. Many of these metabolites contribute to the creation of compatible relations; others behave as elicitors, inducing various protective responses in plants. An important part of the strategy for developing plant resistance against pathogens is the activation of protein synthesis to produce protective ‘pathogenesis-related’ proteins. The family of PR-proteins known to confer the most protective response is chitinases (EC 3.2.1.14, Cht) and β-1,3-glucanases (EC 3.2.1.39, Glu). PR-proteins also include a large multigene family of peroxidases (EC 1.11.1.7, Pod), and increased activity of Pod and expression of the Pod genes leads to the development of resistance to a broad class of pathogens. Despite intensive research on the role of PR-proteins, the question of their participation in the mechanisms of formation of the F.solani–S.tuberosum pathosуstem is not sufficiently studied. Our aim was to investigate the effect of different classes of F. solani metabolites on the activity of chitinase, β-1,3-glucanases and peroxidases in tubers of Solanum tuberosum. Metabolite culture filtrate (CF) and cytoplasmic components were fractionated by extraction of the mycelium with organic solvents, salting out techniques, dialysis, column chromatography and ultrafiltration. Protein, lipid, carbohydrate and polyphenolic fractions of fungal metabolites were derived. Using enzymatic hydrolysis we obtained oligo glycans from fungal cell walls with different molecular weights. The activity of the metabolites was tested using potato tuber discs (d = 16mm, h = 5mm). The activity of PR-proteins of tubers was analyzed in a time course of 2–24 hours. The involvement of the analysed metabolites in the modulation of both early non-specific and late related to pathogenesis reactions was demonstrated. The most effective inducer was isolated from the CF (fraction of total phenolic compounds including naphtazarins). Induction of PR-activity by this fraction was: chitinase - 340-360%, glucanase - 435-450%, soluble forms of peroxidase - 400-560%, related forms of peroxidase - 215-237%. High-inducing activity was observed by the chloroform and acetonitrile extracts of the mycelium (induction of chitinase and glucanase activity was 176-240%, of soluble and bound forms of peroxidase - 190-400%). The fraction of oligo glycans mycelium cell walls of 1.2 kDa induced chitinase and β-1,3-glucanase to 239-320%; soluble forms and related peroxidase to 198-426%. Oligo glycans cell walls of 5-10 kDa had a weak suppressor effect - chitinase (21-25%) and glucanase (25-28%) activity; had no effect on soluble forms of peroxidase, but induced to 250-270% activity related forms. The CF polysaccharides of 8.5 kDa and 3.1 kDa inhibited synchronously the glucanase and chitinase specific response in step (after 24 hours at 42-50%) and the step response induced nonspecific peroxidase activity: soluble forms 4.8 -5.2 times, associated forms 1.4-1.6 times.

Keywords: fusarium solani, PR-proteins, peroxidase, solanum tuberosum

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Authors: Dagmara A. Niedziela, Mark P. Murphy, Orla M. Keane, Finola C. Leonard

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Staphylococcus aureus is the most frequent cause of clinical and subclinical bovine mastitis in Ireland. Mastitis caused by S. aureus is often chronic and tends to recur after antibiotic treatment. This may be due to several virulence factors, including attributes that enable the bacterium to internalize into bovine mammary epithelial cells, where it may evade antibiotic treatment, or evade the host immune response. Four bovine-adapted lineages (CC71, CC97, CC151 and ST136) were identified among a collection of Irish S. aureus mastitis isolates. Genotypic variation of mastitis-causing strains may contribute to different presentations of the disease, including differences in milk somatic cell count (SCC), the main method of mastitis detection. The objective of this study was to investigate the influence of bacterial strain and lineage on host immune response, by employing cell culture methods in vitro as well as an in vivo infection model. Twelve bovine adapted S. aureus strains were examined for internalization into bovine mammary epithelial cells (bMEC) and their ability to induce an immune response from bMEC (using qPCR and ELISA). In vitro studies found differences in a variety of virulence traits between the lineages. Strains from lineages CC97 and CC71 internalized more efficiently into bovine mammary epithelial cells (bMEC) than CC151 and ST136. CC97 strains also induced immune genes in bMEC more strongly than strains from the other 3 lineages. One strain each of CC151 and CC97 that differed in their ability to cause an immune response in bMEC were selected on the basis of the above in vitro experiments. Fourteen first-lactation Holstein-Friesian cows were purchased from 2 farms on the basis of low SCC (less than 50 000 cells/ml) and infection free status. Seven cows were infected with 1.73 x 102 c.f.u. of the CC97 strain (Group 1) and another seven with 5.83 x 102 c.f.u. of the CC151 strain (Group 2). The contralateral quarter of each cow was inoculated with PBS (vehicle). Clinical signs of infection (temperature, milk and udder appearance, milk yield) were monitored for 30 days. Blood and milk samples were taken to determine bacterial counts in milk, SCC, white blood cell populations and cytokines. Differences in disease presentation in vivo between groups were observed, with two animals from Group 2 developing clinical mastitis and requiring antibiotic treatment, while one animal from Group 1 did not develop an infection for the duration of the study. Fever (temperature > 39.5⁰C) was observed in 3 animals from Group 2 and in none from Group 1. Significant differences in SCC and bacterial load between groups were observed in the initial stages of infection (week 1). Data is also being collected on cytokines and chemokines secreted during the course of infection. The results of this study suggest that a strain from lineage CC151 may cause more severe clinical mastitis, while a strain from lineage CC97 may cause mild, subclinical mastitis. Diversity between strains of S. aureus may therefore influence the clinical presentation of mastitis, which in turn may influence disease detection and treatment needs.

Keywords: Bovine mastitis, host immune response, host-pathogen interactions, Staphylococcus aureus

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Authors: Vaibhav D. Bhatt, Anju P. Kunjadia, D. S. Nauriyal, Bhumika J. Joshi, Chaitanya G. Joshi

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Metagenomic analysis of milk samples collected from local cattle breed, kankrej (Bos indicus), Gir (Bos indicus) and Crossbred (Bos indicus X Bos taurus) cattle harbouring subclinical mastitis was carried out by next-generation sequencing (NGS) 454 GS-FLX technology. Around 56 different species including members of Enterobacteriales, Pseudomonadales, Bacillales and Lactobacillales with varying abundance were detected in infected milk. The interesting presence of bacteriophages against Staphylococcus aureus, Escherichia coli, Enterobacter and Yersinia species were observed, especially Enterobacteria and E. coli phages (0∙32%) in Kankrej, Enterobacteria and Staphylococcus phages (1∙05%) in Gir and Staphylococcus phages (2∙32%) in crossbred cattle. NGS findings suggest that phages may be involved in imparting natural resistance of the cattle against pathogens. Further infected milk samples were subjected for bacterial isolation. Fourteen different isolates were identified, and DNA was extracted. Genes (Tet-K, Msr-A, and Mec-A) providing antibiotic resistance to the bacteria were screened by Polymerase Chain Reaction and results were validated with traditional antibiotic assay. Total 3 bacteriophages were isolated from nearby environment of the cattle farm. The efficacy of phages was checked against multi-drug resistant bacteria, identified by PCR. In-vivo study was carried out for phage therapy in mammary glands of female rats “Wister albino”. Mammary glands were infused with MDR isolates for 3 consecutive days. Recovery was observed in infected rats after intramammary infusion of sterile phage suspension. From day 4th onwards, level of C-reactive protein was significant increases up to day 12th . However, significant reduction was observed between days 12th to 18th post treatment. Bacteriophages have significant potential as antibacterial agents and their ability to replicate exponentially within their hosts and their specificity, make them ideal candidates for more sustainable mastitis control.

Keywords: bacteriophages, c-reactive protein, mastitis, metagenomic analysis

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Authors: Amanda Cristina de Oliveira, Elias de Souza Monteiro Filho, Roberta Ceriani

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Liquid-liquid extraction in aqueous two-phase systems (ATPSs) constitutes a powerful tool for purifying bio-materials, such as cells, organelles, proteins, among others. In this work, the extraction of the bovine serum albumin (BSA) has been studied in systems formed by polyethylene glycol (PEG) (1500, 4000, and 6000 g.mol⁻¹) + potassium sodium tartrate + water at 303.15°K. Phase diagrams were obtained by turbidimetry and Merchuk’s method (1998). The experimental tie-lines were described using the Othmer-Tobias and Bancroft correlations. ATPSs were correlated with the nonrandom two-liquid (NRTL) model. The results were considered excellent according to global root-mean-square deviations found which were between 0,72 and 1,13%. The concentrations of the proteins in each phase were determined by spectrophotometry at 280 nm, finding partition efficiencies greater than 71%.

Keywords: aqueous two phases systems, bovine serum albumin , liquid-liquid extraction, polyethylene glycol

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Authors: Intanri Kurniati, Raja Iqbal Mulya Harahap, Agustyas Tjiptaningrum, Reni Zuraida

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Background: Diabetes mellitus is still increasing and has become a health and social burden in the world. It is known that glycation among various proteins is increased in diabetic patients compared with non-diabetic subjects. Some of these glycated proteins are suggested to be involved in the development and progression of chronic diabetic complications. Among these glycated proteins, glycated hemoglobin (HbA1C) is commonly used as the gold standard index of glycemic control in the clinical setting. HbA1C testing has some methods, and the most commonly used is immunoturbidimetry. This research aimed to compare the HbA1c level between immunoturbidimetry and HbA1C level in T2DM patients. Methods: This research involves 77 patients from Abd Muluk Hospital Bandar Lampung; the patient was asked for consent in this research, then underwent phlebotomy to be tested for HbA1C; the sample was then examined for HbA1C with Turbidimetric Inhibition Immunoassay (TINIA) and High-Performance Liquid Chromatography (HPLC) method. Result: Mean± SD of the samples with the TINIA method was 9.2±1,2; meanwhile, the level HbA1C with the HPLC method is 9.6±1,2. The t-test showed no significant difference between the group subjects. (p<0.05). It was proposed that the two methods have high suitability in testing, and both are eligibly used for the patient. Discussion: There was no significant difference among research subjects, indicating that the high conformity of the two methods is suitable to be used for monitoring patients clinically. Conclusion: There is increasing in HbA1C level in a patient with T2DM measured with HPLC and or Turbidimetric Inhibition Immunoassay (TINIA) method, and there were no significant differences among those methods.

Keywords: diabetes mellitus, glycated albumin, HbA1C, HPLC, immunoturbidimetry

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Authors: Prajna Paramita Naik, Sujit Kumar Bhutia

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Background: Regardless of the development multimodal treatment strategies, oral squamous cell carcinoma (OSCC) is often associated with a high rate of recurrence, metastasis and chemo- and radio- resistance. The present study inspected the relevance of CD44, ABCB1 and ADAM17 expression as a putative stem cell compartment in oral squamous cell carcinoma (OSCC) and deciphered the role of autophagy in regulating the expression of aforementioned proteins, stemness and chemoresistance. Methods: A retrospective analysis of CD44, ABCB1 and ADAM17 expression with respect to the various clinicopathological factors of sixty OSCC patients were determined via immunohistochemistry. The correlation among CD44, ABCB1 and ADAM17 expression was established. Sphere formation assay, flow cytometry and fluorescence microscopy were conducted to elucidate the stemness and chemoresistance nature of established cisplatin-resistant oral cancer cells (FaDu). The pattern of expression of CD44, ABCB1 and ADAM17 in parental (FaDu-P) and resistant FaDu cells (FaDu-CDDP-R) were investigated through fluorescence microscopy. Western blot analysis of autophagy marker proteins was performed to compare the status of autophagy in parental and resistant FaDu cell. To investigate the role of autophagy in chemoresistance and stemness, sphere formation assay, immunofluorescence and Western blot analysis was performed post transfection with siATG14 and the level of expression of autophagic proteins, mitochondrial protein and stemness-associated proteins were analyzed. The statistical analysis was performed by GraphPad Prism 4.0 software. p-value was defined as follows: not significant (n.s.): p > 0.05;*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001 were considered statistically significant. Results: In OSCC, high CD44, ABCB1 and ADAM17 expression were significantly correlated with higher tumor grades and poor differentiation. However, the expression of these proteins was not related to the age and sex of OSCC patients. Moreover, the expression of CD44, ABCB1 and ADAM17 were positively correlated with each other. In vitro and OSCC tissue double labeling experiment data showed that CD44+ cells were highly associated with ABCB1 and ADAM17 expression. Further, FaDu-CDDP-R cells showed higher sphere forming capacity along with increased fraction of the CD44+ population and β-catenin expression FaDu-CDDP-R cells also showed accelerated expression of CD44, ABCB1 and ADAM17. A comparatively higher autophagic flux was observed in FaDu-CDDP-R against FaDu-P cells. The expression of mitochondrial proteins was noticeably reduced in resistant cells as compared to parental cells indicating the occurrence of autophagy-mediated mitochondrial degradation in oral cancer. Moreover, inhibition of autophagy was coupled with the decreased formation of orospheres suggesting autophagy-mediated stemness in oral cancer. Blockade of autophagy was also found to induce the restoration of mitochondrial proteins in FaDu-CDDP-R cells indicating the involvement of mitophagy in chemoresistance. Furthermore, a reduced expression of CD44, ABCB1 and ADAM17 was also observed in ATG14 deficient cells FaDu-P and FaDu-CDDP-R cells. Conclusion: The CD44+ ⁄ABCB1+ ⁄ADAM17+ expression in OSCC might be associated with chemoresistance and a putative CSC compartment. Further, the present study highlights the contribution of mitophagy in chemoresistance and confirms the potential involvement of autophagic regulation in acquisition of stem-like characteristics in OSCC.

Keywords: ABCB1, ADAM17, autophagy, CD44, chemoresistance, mitophagy, OSCC, stemness

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Authors: Qingxin Chen, Liben Jiang, Andrew Smith, Karim Hadjri

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Since 2007, the Chinese food industry has undergone serious food contamination in the baby dairy industry, especially milk powder contamination. One of the milk powder products was found to contain melamine and a significant number (294,000) of babies were affected by kidney stones. Due to growing concerns among consumers about food safety and protection, and high pressure from central government, companies must take radical action to ensure food quality protection through the use of an appropriate quality management system. Previously, though researchers have investigated the health and safety aspects of food industries and products, quality issues concerning food products in China have been largely over-looked. Issues associated with baby dairy products and their quality issues have not been discussed in depth. This paper investigates the impact of quality management systems on the Chinese baby dairy product industry. A literature review was carried out to analyse the use of quality management systems within the Chinese milk power market. Moreover, quality concepts, relevant standards, laws, regulations and special issues (such as Melamine, Flavacin M1 contamination) have been analysed in detail. A qualitative research approach is employed, whereby preliminary analysis was conducted by interview, and data analysis based on interview responses from four selected Chinese baby dairy product companies was carried out. Through the analysis of literature review and data findings, it has been revealed that for quality management system that has been designed by many practitioners, many theories, models, conceptualisation, and systems are present. These standards and procedures should be followed in order to provide quality products to consumers, but the implementation is lacking in the Chinese baby dairy industry. Quality management systems have been applied by the selected companies but the implementation still needs improvement. For instance, the companies have to take measures to improve their processes and procedures with relevant standards. The government need to make more interventions and take a greater supervisory role in the production process. In general, this research presents implications for the regulatory bodies, Chinese Government and dairy food companies. There are food safety laws prevalent in China but they have not been widely practiced by companies. Regulatory bodies must take a greater role in ensuring compliance with laws and regulations. The Chinese government must also play a special role in urging companies to implement relevant quality control processes. The baby dairy companies not only have to accept the interventions from the regulatory bodies and government, they also need to ensure that production, storage, distribution and other processes will follow the relevant rules and standards.

Keywords: baby dairy product, food quality, milk powder contamination, quality management system

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Authors: Djeddi Khaled, Houssou Hind, Miloudi Abdellatif, Rabah Siham

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In the field of veterinary medicine, there is a growing application of artificial intelligence (AI) for diagnosing bovine mastitis, a prevalent inflammatory disease in dairy cattle. AI technologies, such as automated milking systems, have streamlined the assessment of key metrics crucial for managing cow health during milking and identifying prevalent diseases, including mastitis. These automated milking systems empower farmers to implement automatic mastitis detection by analyzing indicators like milk yield, electrical conductivity, fat, protein, lactose, blood content in the milk, and milk flow rate. Furthermore, reports highlight the integration of somatic cell count (SCC), thermal infrared thermography, and diverse systems utilizing statistical models and machine learning techniques, including artificial neural networks, to enhance the overall efficiency and accuracy of mastitis detection. According to a review of 15 publications, machine learning technology can predict the risk and detect mastitis in cattle with an accuracy ranging from 87.62% to 98.10% and sensitivity and specificity ranging from 84.62% to 99.4% and 81.25% to 98.8%, respectively. Additionally, machine learning algorithms and microarray meta-analysis are utilized to identify mastitis genes in dairy cattle, providing insights into the underlying functional modules of mastitis disease. Moreover, AI applications can assist in developing predictive models that anticipate the likelihood of mastitis outbreaks based on factors such as environmental conditions, herd management practices, and animal health history. This proactive approach supports farmers in implementing preventive measures and optimizing herd health. By harnessing the power of artificial intelligence, the diagnosis of bovine mastitis can be significantly improved, enabling more effective management strategies and ultimately enhancing the health and productivity of dairy cattle. The integration of artificial intelligence presents valuable opportunities for the precise and early detection of mastitis, providing substantial benefits to the dairy industry.

Keywords: artificial insemination, automatic milking system, cattle, machine learning, mastitis

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Authors: Amin Abbasi, Mahdi Asghari Ozma

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Background and Objective:Enterococcus faecium is a normal flora of the human gastrointestinal tract that causes infection in the host body under conditions such as biofilm formation, in which the use of antibiotics causes changes in these pathogenic mechanisms. In this study, we aimed to evaluate comprehensively the changes in E.faecium when exposed to sub-MIC of the gentamicin,especiallythe biofilm formation rate. Materials and Methods: For this study, the keywords "Enterococcus faecium ", "Biofilm", and "Gentamicin" in the databases PubMed, Google Scholar, Sid, and MagIran between 2015 and 2021 were searched, and 14 articles were chosen, studied, and analyzed. Results: Gentamicin significantly had increased biofilm formation in most of the isolates in the studies. Increased expression of the genes (efaA and esp) and proteins involved in biofilm formation and decreased expression of the genes (gelE and cylA) involved in spreading and proteins involved in metabolism and cell division in E.faecium were the most significant cause of the biofilm formation, which were increased in sub-MIC gentamicin-treated situation. Conclusion: Inadequate use of gentamicin intensify biofilm formation of E.faecium, which can make the treatment of infections caused by this bacterium difficult.

Keywords: biofilm, enterococcus faecium, gentamicin, proteome

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Authors: Zahra Akbari, Farzin Zokaee, Talat Ghomashchi

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Whey is a by-product of the dairy industry whose major components are lactose (44–52 g/L), proteins (6–8 g/L) and mineral salts (4–9 g/L). Approximately 50% of 121 million tons of whey produced in the world in 1993 were disposed into rivers, lakes or other water bodies, treated in wastewater treatment plants or loaded onto land. This represents a significant loss of resources and causes serious pollution problems since whey is a heavy organic pollutant with high COD and BOD values, 40–60 g/L and 50–80 g/L, respectively. The removal of cheese whey proteins and minerals represent an important task both in environmental and in food sciences. The most important treatments which are considered in this study, have been done by using lime, Al2O3, FeCl3 and AlCl3 along with heating and also acidic-alkaline method. Results show that the best way for removal of protein is accomplished with adding HCl to decrease pH from 6 to 4, boiling for 20 min, and filtering protein aggregates. Also partial demineralization in whey solution for reducing ash is accomplished by adding NaOH to increase pH to 7.2 and heating solution for 20 min.

Keywords: whey treatment, dairy industry, precipitation, protein, mineral

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Authors: Nurulhikma Md Isa, Intan Elya Suka, Nur Farhana Roslan, Chew Bee Lynn

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The evolutionarily conserved N-end rule pathway marks proteins for degradation by the Ubiquitin Proteosome System (UPS) based on the nature of their N-terminal residue. Proteins with a destabilizing N-terminal residue undergo a series of condition-dependent N-terminal modifications, resulting in their ubiquitination and degradation. Intensive research has been carried out in Arabidopsis previously. The group VII Ethylene Response Factor (ERFs) transcription factors are the first N-end rule pathway substrates found in Arabidopsis and their role in regulating oxygen sensing. ERFs also function as central hubs for the perception of gaseous signals in plants and control different plant developmental including germination, stomatal aperture, hypocotyl elongation and stress responses. However, nothing is known about the role of this pathway during fruit development and ripening aspect. The plant model system Arabidopsis cannot represent fleshy fruit model system therefore tomato is the best model plant to study. PROTEOLYSIS6 (PRT6) is an E3 ubiquitin ligase of the N-end rule pathway. Two homologs of PRT6 sequences have been identified in tomato genome database using the PRT6 protein sequence from model plant Arabidopsis thaliana. Homology search against Ensemble Plant database (tomato) showed Solyc09g010830.2 is the best hit with highest score of 1143, e-value of 0.0 and 61.3% identity compare to the second hit Solyc10g084760.1. Further homology search was done using NCBI Blast database to validate the data. The result showed best gene hit was XP_010325853.1 of uncharacterized protein LOC101255129 (Solanum lycopersicum) with highest score of 1601, e-value 0.0 and 48% identity. Both Solyc09g010830.2 and uncharacterized protein LOC101255129 were genes located at chromosome 9. Further validation was carried out using BLASTP program between these two sequences (Solyc09g010830.2 and uncharacterized protein LOC101255129) to investigate whether they were the same proteins represent PRT6 in tomato. Results showed that both proteins have 100 % identity, indicates that they were the same gene represents PRT6 in tomato. In addition, we used two different RNAi constructs that were driven under 35S and Polygalacturonase (PG) promoters to study the function of PRT6 during tomato developmental stages and ripening processes.

Keywords: ERFs, PRT6, tomato, ubiquitin

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Authors: P. Joris, E. Lombard, X. Cameleyre, G. Navarro, A. Paillet, N. Gorret, S. E. Guillouet

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Today, on the international space station, multiple supplies are needed per year to supply food and spare parts and to take out waste. But as it is planned to go longer and further into space these supplies will no longer be possible. The astronaut life support system must be able of continuously transform waste into valuable compounds. Two types of production were identified as critical and could be be supplemented by microorganisms. On the one hand, since microgravity causes rapid muscle loss, single cell proteins (SCPs) could be used as protein rich feed or food. On the other hand, having enough building materials to build an advanced habitat will not be possible only by transporting space goods from earth to mars for example. The bacterium Cupriavidus. necator is well known for its ability to produce a large amount of proteins or of polyhydroxyalkanoate biopolymers (PHAs) depending on its implementation. By coupling the life support system to a 3D-printer, astronauts could be supplied with an unlimited amount of building materials. Additionally, based on the design of the life support system, waste streams have been identified: urea from the crew urine and volatile fatty acids (VFAs) from a first stage of organic waste (excrement and food waste) treatment through anaerobic digestion. Thus, the objective of this, within the Spaceship.Fr project, was to demonstrate the feasibility of producing SCPs and PHAs from VFAs and urea in bioreactor. Because life support systems operate continuously as loops, continuous culture experiments were chosen and the effect of the bioreactor dilution rate on biomass composition was investigated. Total transformation of the carbon source into biomass with high SCP or PHA content was achieved in all cases. We will present the transformation performances of VFAs and urea by the bacteria in bioreactor in terms of titers, yields and productivities but also in terms of the quality of SCP and PHA produced, nucleic acid content. We will further discuss the envisioned integration of our process within life support systems.

Keywords: life support system, space travel, waste treatment, single cell proteins, polyhydroxyalkanoates, bioreactor

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Authors: Natapol Pumipuntu

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Staphylococcus argenteus is the emerging species of S. aureus complex. It was generally misidentified as S. aureus by standard techniques and their features. S. argenteus is possibly emerging in both humans and animals, as well as increasing worldwide distribution. The objective of this study was to differentiate and identify S. argenteus from S. aureus, which has been collected and isolated from milk samples of subclinical bovine mastitis cases in Maha Sarakham province, Northeastern of Thailand. Twenty-one isolates of S. aureus, which confirmed by conventional methods and immune-agglutination method were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and multilocus sequence typing (MLST). The result from MALDI-TOF MS and MLST showed 6 from 42 isolates were confirmed as S. argenteus, and 36 isolates were S. aureus, respectively. This study indicated that the identification and classification method by using MALDI-TOF MS and MLST could accurately differentiate the emerging species, S. argenteus, from S. aureus complex which usually misdiagnosed. In addition, the identification of S. argenteus seems to be very limited despite the fact that it may be the important causative pathogen in bovine mastitis as well as pathogenic bacteria in food and milk. Therefore, it is very necessary for both bovine medicine and veterinary public health to emphasize and recognize this bacterial pathogen as the emerging disease of Staphylococcal bacteria and need further study about S. argenteus infection.

Keywords: Staphylococcus argenteus, subclinical bovine mastitis, Staphylococcus aureus complex, mass spectrometry, MLST

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Authors: Panomporn Wisuthseriwong, Hatairuk Tungkasen, Siyaporn Namsongsan, Chanikarn Srinark, Mayuva Youngsabanant-Areekijseree

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The objective of the present study was to evaluate the morphology of porcine cumulus-oocyte complexes (pCOCs) and follicular fluid during follicular development. The samples were obtained from local slaughterhouses in Nakorn Pathom Province, Thailand. Pigs were classified as either in the follicular phase or luteal phase. Porcine follicles (n = 3,510) were categorized as small (1-3 mm in diameters; n=2,910), medium (4-6 mm in diameters; n=530) and large (7-8 mm in diameters; n=70). Then pCOCs and follicular fluid were collected. Finally, we found that the oocytes can be categorized into intact cumulus cells layer oocyte, multi-cumulus cells layer oocyte, partial cumulus cells layer oocyte, completely denuded oocyte and degenerated oocyte. They showed high percentage of intact and multi-cumulus cells layer oocytes from small follicles (54.68%) medium follicles (69.06%) and large follicles (68.57%), which have high potential to develop into matured oocytes in vitro. Protein composition of the follicular fluid was separated by SDS-PAGE technique. The result shows that the protein molecular weight in the small and medium follicles are 23, 50, 66, 75, 92, 100, 132, 163, 225 and >225 kDa. Meanwhile, protein molecular weight in large follicles are 12, 16, 23, 50, 66, 75, 92, 100, 132, 163, 225 and >225 kDa. All proteins play an important role in promotion and regulation on development, maturation of oocytes and regulation of ovulation. We conclude that the results of discovery can be used porcine secretion proteins for supplement in IVM/IVF technology. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: porcine follicles, porcine oocyte, follicular fluid, SDS-PAGE

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Authors: Katerina H. Takova, Ivan N. Minkov, Gergana G. Zahmanova

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Molecular farming is the production of recombinant proteins in plants with the aim to use the protein as a purified product, crude extract or directly in the planta. Plants gain more attention as expression systems compared to other ones due to the cost effective production of pharmaceutically important proteins, appropriate post-translational modifications, assembly of complex proteins, absence of human pathogens to name a few. In addition, transient expression in plant leaves enables production of recombinant proteins within few weeks. Hepatitis E virus (HEV) is a causative agent of acute hepatitis. HEV causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. Presently, all efforts for development of Hepatitis E vaccine are focused on the Open Read Frame 2 (ORF2) capsid protein as it contains epitopes that can induce neutralizing antibodies. For our purpose, we used the CMPV-based vector-pEAQ-HT for transient expression of HEV ORF2 in Nicotiana benthamina. Different molecular analysis (Western blot and ELISA) showed that HEV ORF2 capsid protein was expressed in plant tissue in high-yield up to 1g/kg of fresh leaf tissue. Electron microscopy showed that the capsid protein spontaneously assembled in low abundance virus-like particles (VLPs), which are highly immunogenic structures and suitable for vaccine development. The expressed protein was recognized by both human and swine HEV positive sera and can be used as a diagnostic reagent for the detection of HEV infection. Production of HEV capsid protein in plants is a promising technology for further HEV vaccine investigations. Here, we reported for a rapid high-yield transient expression of a recombinant protein in plants suitable for vaccine production as well as a diagnostic reagent. Acknowledgments -The authors’ research on HEV is supported with grants from the Project PlantaSYST under the Widening Program, H2020 as well as under the UK Biotechnological and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant ‘Understanding and Exploiting Plant and Microbial Secondary Metabolism’ (BB/J004596/1). The authors want to thank Prof. George Lomonossoff (JIC, Norwich, UK) for his contribution.

Keywords: hepatitis E virus, plant molecular farming, transient expression, vaccines

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Authors: Sumeyra Sevim, Gulsum Gizem Topal, Mercan Merve Tengilimoglu-Metin, Mevlude Kizil

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Aflatoxin M1 (AFM1) is a major toxic and carcinogenic molecule in milk and milk products. Therefore, it poses a risk for public health. Probiotics can be biological agent to remove AFM1. The aim of this study is to evaluate the effect of probiotic bacteria on AFM1 detoxification in phosphate buffer saline. The PBS samples artificially contaminated with AFM1 at concentration 100 pg/ml were prepared with probiotics bacteria that including monoculture (L. plantarum, B. bifidum ATCC, B. animalis ATCC 27672) and binary culture (L. bulgaricus + S. thermophiles, B. bifidum ATCC + B. animalis ATCC 27672, L. plantarum+B. bifidum ATCC, L. plantarum+ B. animalis ATCC 27672). The samples were incubated at 37°C for 4 hours and stored for 1, 5 and 10 days. The toxin was measured by the ELISA. The highest levels of AFM1 binding ability (63.6%) in PBS were detected yoghurt starter bacteria, while L. plantarum had the lowest levels of AFM1 binding ability (35.5%) in PBS. In addition, it was found that there was significant effect of storage on AFM1 binding ability in all groups except the one including B. animalis (p < 0.05). Consequently, results demonstrate that AFM1 detoxification by probiotic bacteria has a potential application to reduce toxin concentrations in yoghurt. Moreover, probiotic strains can react with itself as synergic or antagonist.

Keywords: aflatoxin M1, ELISA, probiotics, storage

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Authors: Juliana Lukša, Bazilė Ravoitytė, Elena Servienė, Saulius Serva

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Totiviridae L-A virus is a persistent Saccharomyces cerevisiae dsRNA virus. It encodes the major structural capsid protein Gag and Gag-Pol fusion protein, responsible for virus replication and encapsulation. These features also enable the copying of satellite dsRNAs (called M dsRNAs) encoding a secreted toxin and immunity to it (known as killer toxin). Viral capsid pore presumably functions in nucleotide uptake and viral mRNA release. During cell division, sporogenesis, and cell fusion, the virions remain intracellular and are transferred to daughter cells. By employing high throughput RNA sequencing data analysis, we describe the influence of solely L-A virus on the expression of genes in three different S. cerevisiae hosts. We provide a new perception into Totiviridae L-A virus-related transcriptional regulation, encompassing multiple bioinformatics analyses. Transcriptional responses to L-A infection were similar to those induced upon stress or availability of nutrients. It also delves into the connection between the cell metabolism and L-A virus-conferred demands to the host transcriptome by uncovering host proteins that may be associated with intact virions. To better understand the virus-host interaction, we applied differential proteomic analysis of virus particle-enriched fractions of yeast strains that harboreither complete killer system (L-A-lus and M-2 virus), M-2 depleted orvirus-free. Our analysis resulted in the identification of host proteins, associated with structural proteins of the virus (Gag and Gag-Pol). This research was funded by the European Social Fund under the No.09.3.3-LMT-K-712-19-0157“Development of Competences of Scientists, other Researchers, and Students through Practical Research Activities” measure.

Keywords: totiviridae, killer virus, proteomics, transcriptomics

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Authors: Sawale Pravin Digambar, G. R. Patil, Shaik Abdul Hussain

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Terminalia arjuna (TA), an important medicinal plant in Indian System of Medicine, is specifically recognized for its recuperative effect on heart ailments. Alcoholic extract of TA (both free and encapsulated) was incorporated into milk to obtain functional dairy beverages. The respective beverages were appropriately flavored and optimized using response surface methodology to improve the sensory appeal. The beverages were evaluated for their compositional, anti-oxidative and various other physico-chemical aspects. Addition of herb (0.3%) extract to flavoured dairy drink (Drink 1) resulted in significantly lowered (p>0.05) HMF content and increased antioxidant activity, total phenol content as compared with control (Control 1). Subsequently, a significant (p>0.05) increase in acidity and sedimentation was also observed. Encapsulated herb (1.8%) incorporated drink (Drink 2) had significantly (P>0.05) enhanced HMF value and decreased antioxidant activity, phenol content as compared to herb added vanilla chocolate dairy drink (Drink 1). It can be concluded that addition of encapsulated TA extract and non-encapsulated TA extract to chocolate dairy drink at 0.3% concentration altered the functional properties vanilla chocolate dairy drink which could be related to the interaction of herb components such as polyphenol with milk protein or maltodextrin/ gum Arabic matrix.

Keywords: Terminalia arjuna, encapsulate, antioxidant activity, physicochemical study

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