Search results for: protein isolates

Authors: Daina Jonkus, Solvita Petrovska, Dace Smiltina, Lasma Cielava

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The beta-lactoglobulin and kappa-casein are milk proteins which are important for milk composition. Cows with beta-lactoglobulin and kappa-casein gene BB genotypes have highest milk crude protein and fat content. The aim of the study was to determinate the frequencies of milk protein gene polymorphisms in local Latvian Brown (LB) cows breed and analyze the influence of beta-lactoglobulin and kappa-casein genotypes to milk productivity traits. 102 cows’ genotypes of milk protein genes were detected using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) and electrophoresis on 3% agarose gel. For beta-lactoglobulin were observed 2 types of alleles A and B and for kappa-casein 3 types: A, B and E. Highest frequency in beta-lactoglobulin gene was observed for B allele – 0.926. Molecular analysis of beta-lactoglobulin gene shows 86.3% of individuals are homozygous by B allele and animals are with genotypes BB and 12.7% of individuals are heterozygous with genotypes AB. The highest milk yield 4711.7 kg was for 1st lactation cows with AB genotypes, whereas the highest milk protein content (3.35%) and fat content (4.46 %) was for BB genotypes. Analysis of the kappa-casein locus showed a prevalence of the A allele – 0.750. The genetic variant of B was characterized by a low frequency – 0.240. Moreover, the frequency of E occurred in the LB cows’ population with very low frequency – 0.010. 54.9 % of cows are homozygous with genotypes AA, and only 4.9 % are homozygous with genotypes BB. 32.8 % of individuals are heterozygous with genotypes AB, and 2.0 % are with AE. The highest milk productivity was for 1st lactation cows with AB genotypes: milk yield 4620.3 kg, milk protein content 3.39% and fat content 4.53 %. According to the results, in local Latvian brown there are only 2.9% of cows are with BB-BB genotypes, which is related to milk coagulation ability and affected cheese production yield. Acknowledgment: the investigation is supported by VPP 2014-2017 AgroBioRes Project No. 3 LIVESTOCK.

Keywords: beta-lactoglobulin, cows, genotype frequencies, kappa-casein

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Authors: Purva Mishra, Rajesh Potlia, Kuljeet Singh Sandhu

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The role of glycyl residues in the protein structure has lingered within the research community for the last several decades. Glycyl residue is the only amino acid that is achiral due to the lack of a side chain and can, therefore, exhibit Ramachandran conformations that are disallowed for L-amino acids. The structural and functional significance of glycyl residues with L-disallowed conformation, however, remains obscure. Through statistical analysis of various datasets, we found that the glycyls with L-disallowed conformations are over-represented at disease-associated sites and tend to be evolutionarily conserved. The mutations of L-disallowed glycyls tend to destabilize the native conformation, reduce protein solubility, and promote inter-molecular aggregations. We uncovered a structural motif referred to as “β-crescent” formed around the L-disallowed glycyl, which prevents β-sheet aggregation by disrupting the alternating pattern of β-pleats. The L-disallowed conformation of glycyls also holds predictive power to infer the pathogenic missense variants. Altogether, our observations highlight that the L-disallowed conformation of glycyls is selected to facilitate native folding and prevent inter-molecular aggregations. The findings may also have implications for designing more stable proteins and prioritizing the genetic lesions implicated in diseases.

Keywords: Ramachandran plot, β-sheet, protein stability, protein aggregation

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Authors: Mohamed M. Alsull

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In the present study, two microalgae species “Nannochloropsis sp. and Tetraselmis sp.” isolated from Penang National Park coastal waters, Malaysia; were cultivated under combined various laboratory conditions “salinity, light, nitrogen limitation and starvation”. Growth rate, dry weight, chlorophyll a content, total lipid and protein contents, were estimated at mid exponential growth phase. Both Nannochloropsis sp. and Tetraselmis sp. showed remarkable decrease in growth rate, chlorophyll a content and protein content companied with increase in lipid content under nitrogen limitation and starvation conditions. Maintaining Nannochloropsis sp. under salinity 15‰ caused only significant decrease in total protein content; while Tetraselmis sp. grown at the same salinity caused decrease in the growth rate, chlorophyll a, dry weight and total protein content only when nitrogen was available.

Keywords: biochemical composition, light, microalgae, nitrogen limitation, salinity

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Authors: Sena Su, Burak Ozbek, Yesim M. Sahin, Sevil Yucel, Dilek Kazan, Faik N. Oktar, Nazmi Ekren, Oguzhan Gunduz

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In recent years, protein, lipid or polysaccharide-based polymers have been used in order to develop biodegradable materials and their chemical nature determines the physical properties of the resulting films. Among these polymers, proteins from different sources have been extensively employed because of their relative abundance, film forming ability, and nutritional qualities. In this study, the biodegradable composite nanofiber films based on fish sarcoplasmic protein (FSP) were prepared via electrospinning technique. Biodegradable polycaprolactone (PCL) was blended with the FSP to obtain hybrid FSP/PCL nanofiber mats with desirable physical properties. Mixture solutions of FSP and PCL were produced at different concentrations and their density, viscosity, electrical conductivity and surface tension were measured. Mechanical properties of electrospun nanofibers were evaluated. Morphology of composite nanofibers was observed using scanning electron microscopy (SEM). Moreover, Fourier transform infrared spectrometer (FTIR) studies were used for analysis chemical composition of composite nanofibers. This study revealed that the FSP based nanofibers have the potential to be used for different applications such as biodegradable packaging, drug delivery, and wound dressing, etc.

Keywords: edible film, electrospinning, fish sarcoplasmic protein, nanofiber

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Authors: Hawbash Jalil

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In this study, the effects of whey protein based films on various properties of kashar cheese were examined. In the study, edible film solutions based on whey protein isolate, whey protein isolate + transglutaminase enzyme and whey protein isolate + chitosan were produced and Kashar cheese samples were coated with these films by dipping method and stored at +4 ºC for 60 days. Chemical, microbiological and textural analyzes were carried out on samples at 0, 30 and 60 days of storage. As a result of the study, the highest dry matter and total nitrogen values were obtained from uncoated control samples This is an indication that the coatings limit water vapor permeability. The highest acidity and pH values obtained from the samples as storage results were 3.33% and 5.86%, respectively, in the control group samples. Both acidity and pH rise in these groups, is a consequence of the buffering of pH changes of hydrolsis products which are as a result of proteolysis occurring in the sample. Nitrogen changes and lipolysis values, which are indicative of the degree of hydrolysis of proteins and triglycerides in kashar cheese, were generally higher in the control group This result is due to limiting the micro organism reproduction by limiting the gas passage of the coatings. Hardness and chewiness values of the textural properties of the samples were significantly reduced in uncoated control samples compared to the coated samples due to maturation. The chitosan film coatings used in the study limited the development of mold yeast until the 30th day but after that did not yield successful results in this respect.

Keywords: chitosan, edible film, transglutaminase, whey protein

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Authors: Mohammad Javad Esmaeili

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Background: Coagulopathy is one of the complications with end stage renal disease with high prevalence in the world. Thromboelastogram is adynamic test for evaluation of coagulopathy and we have compared our patient's coagulation profiles with the results of TEG. Material and methods: In this study 50 patients with ESRD who were on regular hemodialysis for at least 6 months was selected with simple sampling and their coagulation profile was done with blood sampling and also TEG was done for every patient. Data were analyzed with SPSS and P<0.05 consider significant. Results: Protein s, Protein c and Antithrombin III deficiency was detected in 32%, 16% and 20% of patients and activated protein c resistance was abnormal in 2% of patients. In TEG, R time in 49% and K in 22/5% of patients was lower than normal and a-angle in 26% and maximum amplitude in 36% of patients was upper than normal (Hypercoagulable state). PS with R and ATIII with K have correlation. Conclusion: R time and K in TEG can be a suitable screening test in patients with suspicious to PS and ATIII deficiency.

Keywords: thromboelastography, chronic kidney disease, Coagulating disorder, hemodialysis

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Authors: Naheed Niaz, Nimra Naeem, Bushra Uzair, Riffat Tahira

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Antibacterial activity of different traditional plants essential oils against clinical isolates of Methicillin-resistant Staphylococcus aureus (MRSA) through disk diffusion method was evaluated. All the tested essential oils, in different concentrations, inhibited growth of S. aureus to varying degrees. Cinnamon and Thyme essential oils were observed to be the “best” against test pathogen. Even at lowest concentration of these essential oils i.e. 25 µl/ml, clear zone of inhibition was recorded 9+0.085mm and 8+0.051mm respectively, and at higher concentrations there was a total reduction in growth of MRSA. The study also focused on analyzing the synergistic effects of essential oils in combination with amoxicillin. Results showed that oregano and pennyroyal mint essential oils which were not very effective alone turned out to be strong synergistic enhancers. The activity increased with increase in concentration of the essential oils. It may be concluded from present results that cinnamon and thyme essential oils could be used as potential antimicrobial source for the treatment of infections caused by Methicillin-resistant Staphylococcus aureus (MRSA).

Keywords: Staphylococcus aureus, essential oils, antibiotics, combination therapy, minimum inhibitory concentration

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Authors: Siddharth Deshpande, Nihar Masurkar, Vallerinteavide Mavelli Girish, Malan Desai, Chester Drum

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Folding and stabilization of recombinant proteins remain a consistent challenge for industrial and therapeutic applications. Proteins derived from thermophilic bacteria often have superior expression and stability qualities. To develop a generalizable approach to protein folding and stabilization, we tested the hypothesis that wrapping a thermostable exoshell around a protein substrate would aid folding and impart thermostable qualities to the internalized substrate. To test the effect of internalizing a protein within a thermostable exoshell (tES), we tested in vitro folding and stability using green fluorescent protein (GFPuv), horseradish peroxidase (HRP) and renilla luciferase (rLuc). The 8nm interior volume of a thermostable ferritin assembly was engineered to accommodate foreign proteins and either present a positive, neutral or negative interior charge environment. We further engineered the tES complex to reversibly assemble and disassemble with pH titration. Template proteins were expressed as inclusion bodies and an in vitro folding protocol was developed that forced proteins to fold inside a single tES. Functional yield was improved 100-fold, 100-fold and 150-fold with use of tES for GFPuv, HRP and rLuc respectively and was highly dependent on the internal charge environment of the tES. After folding, functional proteins could be released from the tES folding cavity using size exclusion chromatography at pH 5.8. Internalized proteins were tested for improved stability against thermal, organic, urea and guanidine denaturation. Our results demonstrated that thermostable exoshells can efficiently refold and stabilize inactive aggregates into functional proteins.

Keywords: thermostable shell, in vitro folding, stability, functional yield

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Authors: Lei Wang, Yingzhou Ni, Amr M. Bakry, Hao Cheng, Li Liang

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Food proteins have been widely used as carrier materials because of their multiple functional properties. In this study, alfa-tocopherol was encapsulated in the oil phase of an oil-in-water emulsion stabilized with whey protein isolate (WPI). The influence of WPI concentration and resveratrol or ascorbic acid on the decomposition of alfa-tocopherol in the emulsion during storage is discussed. Decomposition decreased as WPI concentrations increased. Decomposition was delayed at ascorbic acid/WPI molar ratios lower than 5 but was promoted at higher ratios. Resveratrol partitioned into the oil-water interface by binding to WPI and its cis-isomer is believed to have contributed most of the protective effect of this polyphenol. These results suggest the possibility of using the emulsifying and ligand-binging properties of WPI to produce carriers for simultaneous encapsulation of alfa-tocopherol and resveratrol in a single emulsion system.

Keywords: stability, alfa-tocopherol, resveratrol, whey protein isolate

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Authors: Akanksha Agrawal, Richa Rathor, Geetha Suryakumar

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Muscle represents about ¾ of the body mass, and a healthy muscular system is required for human performance. A healthy muscular system is dynamically balanced via the catabolic and anabolic process. High altitude associated hypoxia altered this redox balance via producing reactive oxygen and nitrogen species that ultimately modulates protein structure and function, hence, disrupts proteostasis or protein homeostasis. The mechanism by which proteostasis is clinched includes regulated protein translation, protein folding, and protein degradation machinery. Perturbation in any of these mechanisms could increase proteome imbalance in the cellular processes. Altered proteostasis in skeletal muscle is likely to be responsible for contributing muscular atrophy in response to hypoxia. Therefore, we planned to elucidate the mechanism involving altered proteostasis leading to skeletal muscle atrophy under chronic hypobaric hypoxia. Material and Methods-Male Sprague Dawley rats weighing about 200-220 were divided into five groups - Control (Normoxic animals), 1d, 3d, 7d and 14d hypobaric hypoxia exposed animals. The animals were exposed to simulated hypoxia equivalent to 282 torr pressure (equivalent to an altitude of 7620m, 8% oxygen) at 25°C. On completion of chronic hypobaric hypoxia (CHH) exposure, rats were sacrificed, muscle was excised and biochemical, histopathological and protein synthesis signaling were studied. Results-A number of changes were observed with the CHH exposure time period. ROS was increased significantly on 07 and 14 days which were attributed to protein oxidation via damaging muscle protein structure by oxidation of amino acids moiety. The oxidative damage to the protein further enhanced the various protein degradation pathways. Calcium activated cysteine proteases and other intracellular proteases participate in protein turnover in muscles. Therefore, we analysed calpain and 20S proteosome activity which were noticeably increased at CHH exposure as compared to control group representing enhanced muscle protein catabolism. Since inflammatory markers (myokines) affect protein synthesis and triggers degradation machinery. So, we determined inflammatory pathway regulated under hypoxic environment. Other striking finding of the study was upregulation of Akt/PKB translational machinery that was increased on CHH exposure. Akt, p-Akt, p70 S6kinase, and GSK- 3β expression were upregulated till 7d of CHH exposure. Apoptosis related markers, caspase-3, caspase-9 and annexin V was also increased on CHH exposure. Conclusion: The present study provides evidence of disrupted proteostasis under chronic hypobaric hypoxia. A profound loss of muscle mass is accompanied by the muscle damage leading to apoptosis and cell death under CHH. These cellular stress response pathways may play a pivotal role in hypobaric hypoxia induced skeletal muscle atrophy. Further research in these signaling pathways will lead to development of therapeutic interventions for amelioration of hypoxia induced muscle atrophy.

Keywords: Akt/PKB translational machinery, chronic hypobaric hypoxia, muscle atrophy, protein degradation

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Authors: Jayson Wau, David Timi, Anthony Harakuwe, Bruce Bowden, Cherie Motti, Harry Sakulas, Rag Gubag-Sipou

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The latex of F. botryocarpa fruit is applied on sores, wounds and other skin infections in Papua New Guinea ethnotherapeutic practices. Systematic bioassay guided separation and isolation of subsequent fractions of latex extracts resulted in three bioactive fractions active against Staphylococcus aureus and Escherichia coli. This study reports structural elucidation of the three isolates. Structures were determined by physical (M.pt and Rf values) and spectroscopic (1D-1H NMR, 2D-HSQC NMR, 2D-HMBC NMR) and MS ESI-POS. The two methylene protons (2H-1) and (2H-3) resonate as triplets at δ 3.59 and δ 4.99 respectively. Electron dense δ 4.99 (2H-3) on (C-3) depicts the strong electron-withdrawing component, quaternary nitrogen (=N= +). Protons resonating at δ 3.88 and 3.89 are singlets depicting two methoxy groups. Both δ 3.88 and δ 3.89 are para-aryls substituents. The methines δ 9.13 and 8.60 are singlets depicting two lone protons on the indolizidinium aryl component. All isolates, (1), (2) and (3) were identified to be ficuseptine by comparing 1D-NMR assignments. 2D-NMR and MS of (2) found it to be ficuseptine chloride '2, 3-dihydro-6, 8-bis (4-methoxyphenyl)-, 1H-indolizinium chloride'. Their counter ions of the ficuseptines were not established and provide promising lead for the further investigation.

Keywords: Ficus botryocarpa, antimicrobial activity, ficuseptine, sores

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Authors: S. Ibrahim, U. B. Abubakar, S. Danbirni, A. Usman, F. M. Ballah, C. A. Kudi, L. Lawson, G. H. Abdulrazak, I. A. Abdulkadir

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Performing culture and characterization of mycobacteria in low resource settings like Nigeria is a very difficult task to undertake because of the very few and limited laboratories carrying out such an experiment; this is a largely due to stringent and laborious nature of the tests. Hence, a rapid, simple and accurate test for characterization is needed. The “SD BIOLINE TB Ag MPT 64 Rapid ®” is a simple and rapid immunochromatographic test used in differentiating Mycobacteria into Mycobacterium tuberculosis (NTM). The 100 sputa were obtained from patients suspected to be infected with tuberculosis and presented themselves to hospitals for check-up and treatment were involved in the study. The samples were cultured in a class III Biosafety cabinet and level III biosafety practices were followed. Forty isolates were obtained from the cultured sputa, and there were identified as Acid-fast bacilli (AFB) using Zeihl-Neelsen acid-fast stain. All the isolates (AFB positive) were then subjected to the SD BIOLINE Analyses. A total of 31 (77.5%) were characterized as MTBC, while nine (22.5%) were NTM. The total turnaround time for the rapid assay was just 30 minutes as compared to a few days of phenotypic and genotypic method. It was simple, rapid and reliable test to differentiate MTBC from NTM.

Keywords: culture, mycobacteria, non tuberculous mycobacterium, SD Bioline

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Authors: Iftikhar Tayubi, Tabrej Khan, Rayan Alsulmi, Abdulrahman Labban

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Spider produces a special kind of substance. This special kind of substance is called a toxin. The toxin is composed of many types of protein, which differs from species to species. Spider toxin consists of several proteins and non-proteins that include various categories of toxins like myotoxin, neurotoxin, cardiotoxin, dendrotoxin, haemorrhagins, and fibrinolytic enzyme. Protein Sequence information with references of toxins was derived from literature and public databases. From the previous findings, the Spider toxin would be the best choice to treat different types of tumors and cancer. There are many therapeutic regimes, which causes more side effects than treatment hence a different approach must be adopted for the treatment of cancer. The combinations of drugs are being encouraged, and dramatic outcomes are reported. Spider toxin is one of the natural cytotoxic compounds. Hence, it is being used to treat different types of tumors; especially its positive effect on breast cancer is being reported during the last few decades. The efficacy of this database is that it can provide a user-friendly interface for users to retrieve the information about Spiders, toxin and toxin protein of different Spiders species. SPIDTOXD provides a single source information about spider toxins, which will be useful for pharmacologists, neuroscientists, toxicologists, medicinal chemists. The well-ordered and accessible web interface allows users to explore the detail information of Spider and toxin proteins. It includes common name, scientific name, entry id, entry name, protein name and length of the protein sequence. The utility of this database is that it can provide a user-friendly interface for users to retrieve the information about Spider, toxin and toxin protein of different Spider species. The database interfaces will satisfy the demands of the scientific community by providing in-depth knowledge about Spider and its toxin. We have adopted the methodology by using A MySQL and PHP and for designing, we used the Smart Draw. The users can thus navigate from one section to another, depending on the field of interest of the user. This database contains a wealth of information on species, toxins, and clinical data, etc. This database will be useful for the scientific community, basic researchers and those interested in potential pharmaceutical Industry.

Keywords: siptoxd, php, mysql, toxin

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Authors: Sihame Amakrane, Zineb Ouahdi, Mohammed Salah, Said Belaaouad

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\This research explores the efficacy of novel pyrimidine derivatives on bacterial strains such as Escherichia coli, Staphylococcus aureus, and Myccobacterium tuberculosis, utilizing bending energy calculations. Of the 25 compounds examined, 13 displayed potent activity against all the bacterial strains under study, exhibiting bending energy measurements between -7.4 and -10.7 kcal/mol. The -7.4 kcal/mol value corresponds to the bending energy of the SA12 and SA13 compounds with the 2xct protein (Staphylococcus aureus), whereas the -10.7 kcal/molis linked with the bending energy of SA6 and SA11 compounds with the 6GAV protein (Myccobacterium tuberculosis). Further research will involve a QSAR (Quantitative Structure-Activity Relationship) study aimed at constructing a reliable model to combat the aforementioned bacterial strains and a molecular dynamics study to evaluate the stability of ligand-protein complexes.

Keywords: docking, QSAR, bending energy, e. coli

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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: Baharuddin Patandjengi, A. Pabborong, T. Kuswinanti

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Bacterial ring rot caused by a gram-positive Coryneform bacterium Corynebacterium michiganensis subsp. sepedonicus is an important disease on potato crops in the world. The disease still belongs to an A1 quarantine pathogen in Indonesia, although it was found in West Java since 2013. The objective of this study was to know the presence of bacterial ring rot in four potato district areas in South Sulawesi. Infected samples were collected from potato fields and storage warehouses in Enrekang, Gowa, Jeneponto and Bantaeng districts. Potato tuber samples were cut and observed their vasiculer vessels and the bacterial ooze was used for isolation on Nutrient Agar and Nutrient Broth–Yeast extract medium. Bacterial isolates were then morphologically and physiologically characterized. A patogenicity test on eggplant and molecular characterization using PCR with specific primer for Cms (50F and Cms 50 R) was revealed for further identification. The results showed that Cms has become widespread in four districts of South Sulawesi. The bacterial ringrot disease incidence in these districts was reached above 30 %. All of 14 bacterial isolates that identified before using standard methods of EPPO, showed DNA band in size of 224 bp in PCR test, which indicated positively belong to C. michiganensis subsp. sepedonicus.

Keywords: bacterial ring rot, clavibacter michiganensis pv. sepedonicus, PCR, potato

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Authors: Mayank, Navneet Kaur, Narinder Singh

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The V599 mutations in the BRAF protein are extremely oncogenic, responsible for countless of malignant conditions. Along with wild type, V599E, V599D, and V599R are the important mutated variants of the BRAF proteins. The BRAF inhibitory anticancer agents are continuously developing, and sorafenib is a BRAF inhibitor that is under clinical use. The crystal structure of sorafenib bounded to wild type, and V599 is known, showing a similar interaction pattern in both the case. The mutated 599th residue, in both the case, is also found not interacting directly with the co-crystallized sorafenib molecule. However, the IC50 value of sorafenib was found extremely different in both the case, i.e., 22 nmol/L for wild and 38 nmol/L for V599E protein. Molecular docking study and MMGBSA binding energy results also revealed a significant difference in the binding pattern of sorafenib in both the case. Therefore, to explore the role of distinctively situated 599th residue, we have further conducted comprehensive computational studies. The molecular dynamics simulation, residue interaction network (RIN) analysis, and residue correlation study results revealed the importance of the 599th residue on the therapeutic outcome and overall dynamic of the BRAF protein. Therefore, although the position of 599th residue is very much distinctive from the ligand-binding cavity of BRAF, still it has exceptional control over the overall functional outcome of the protein. The insight obtained here may seem extremely important and guide us while designing ideal BRAF inhibitory anticancer molecules.

Keywords: BRAF, oncogenic, sorafenib, computational studies

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Authors: Abdul Matin, Muazzama Akhtar, Shahid Nisar, Saddaf Mazzar, Umer Rashid

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Antibiotic resistance is an increasing concern worldwide now a day. Neisseria gonorrhea and Staphylococcus aureus are known to cause major human sexually transmitted and respiratory diseases respectively. Nanotechnology is an emerging discipline and its application in various fields especially in medical sciences is gigantic. In the present study, we synthesized multi-walled carbon nanotubes (MWNTs) using acid oxidation method and solubilized MWNTs were with length predominantly >500 nm and diameters ranging from 40 to 50 nm. The locally synthesized MWNTs were used against gram positive and negative bacteria to determine their impact on bacterial growth. Clinical isolates of Neisseria gonorrhea (isolate: 4C-11) and Staphylococcus aureus (isolate: 38541) were obtained from local hospital and normally cultured in LB broth at 37°C. Both clinical strains can be obtained on request from University of Gujarat. Spectophometric assay was performed to determine the impact of MWNTs on bacterial growth in vitro. To determine the effect of MWTNs on test organisms, various concentration of MWNTs were used and recorded observation on various time intervals to understand the growth inhibition pattern. Our results demonstrated that MWNTs exhibited toxic effects to Staphylococcus aureus while showed very limited growth inhibition to Neisseria gonorrhea, which suggests the resistant potential of Neisseria against nanoparticles. Our results clearly demonstrate the gradual decrease in bacterial numbers with passage of time when compared with control. Maximum bacterial inhibition was observed at maximum concentration (50 µg/ml). Our future work will include further characterization and mode of action of our locally synthesized MWNTs. In conclusion, we investigated and reported for the first time the inhibitory potential of locally synthesized MWNTs on local clinical isolates of Staphylococcus aureus and Neisseria gonorrhea.

Keywords: antibacterial activity, multi walled carbon nanotubes, Neisseria gonorrhea, spectrophotometer assay, Staphylococcus aureus

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Authors: Pukhrambam Helena Chanu, Janardan Yadav

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This study investigates the potential of various fungal isolates to solubilize zinc and counteract rice pathogens, with the aim of mitigating zinc deficiency and disease prevalence in rice farming. Soil samples from the rhizosphere were collected, and zinc-solubilizing fungi were isolated and purified. Molecular analysis identified Talaromyces sp, Talaromyces versatilis, Talaromyces pinophilus, and Aspergillus terreus as effective zinc solubilizers. Through qualitative and quantitative assessments, it was observed that solubilization efficiencies varied among the isolates over time, with Talaromyces versatilis displaying the highest capacity for solubilization. This variability in solubilization rates may be attributed to differences in fungal metabolic activity and their ability to produce organic acids that facilitate zinc release from insoluble sources in the soil. In inhibition assays against rice pathogens, the fungal isolates exhibited antagonistic properties, with Talaromyces versatilis demonstrating the most significant inhibition rates. This antagonistic activity may be linked to the production of secondary metabolites, such as antibiotics or lytic enzymes by fungi, which inhibit the growth of rice pathogens. The ability of Talaromyces versatilis to outperform other isolates in both zinc solubilization and pathogen inhibition highlights its potential as a multifunctional biocontrol agent in rice cultivation systems. These findings emphasize the potential of fungi as natural solutions for enhancing zinc uptake and managing diseases in rice cultivation. Utilizing indigenous zinc-solubilizing fungi offers a sustainable and environmentally friendly approach to addressing zinc deficiency in soils, reducing the need for chemical fertilizers. Moreover, harnessing the antagonistic activity of these fungi can contribute to integrated disease management strategies, minimizing reliance on synthetic pesticides and promoting ecological balance in agroecosystems. Additionally, the study included the evaluation of dipping time under different concentrations, viz.,10 ppm, 20 ppm, and 30 ppm of biosynthesized nano ZnO on rice seedlings. This investigation aimed to optimize the application of nano ZnO for efficient zinc uptake by rice plants while minimizing potential risks associated with excessive nanoparticle exposure. Evaluating the effects of varying concentrations and dipping durations provides valuable insights into the safe and effective utilization of nano ZnO as a micronutrient supplement in rice farming practices.

Keywords: biosynthesized nano ZnO, rice, root dipping, zinc solubilizing fungi.

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Authors: Olubukayo-Opeyemi Oyetayo, Oscar Mendez-Lucio, Andreas Bender, Hans Kiefer

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The thermal melting curve of a protein provides information on its conformational stability and could provide cues on its aggregation behavior. Naturally-occurring osmolytes have been shown to improve the thermal stability of most proteins in a concentration-dependent manner. They are therefore commonly employed as additives in therapeutic protein purification and formulation. A number of intertwined and seemingly conflicting mechanisms have been put forward to explain the observed stabilizing effects, the most prominent being the preferential exclusion mechanism. We attempted to probe and summarize molecular mechanisms for thermal stabilization of a monoclonal antibody (mAb) by developing quantitative structure-activity relationships using a rationally-selected library of 120 osmolyte-like compounds in the polyhydric alcohols, amino acids and methylamines classes. Thermal stabilization potencies were experimentally determined by thermal shift assays based on differential scanning fluorimetry. The cross-validated QSAR model was developed by partial least squares regression using descriptors generated from Molecular Operating Environment software. Careful evaluation of the results with the use of variable importance in projection parameter (VIP) and regression coefficients guided the selection of the most relevant descriptors influencing mAb thermal stability. For the mAb studied and at pH 7, the thermal stabilization effects of tested compounds correlated positively with their fractional polar surface area and inversely with their fractional hydrophobic surface area. We cannot claim that the observed trends are universal for osmolyte-protein interactions because of protein-specific effects, however this approach should guide the quick selection of (de)stabilizing compounds for a protein from a chemical library. Further work with a large variety of proteins and at different pH values would help the derivation of a solid explanation as to the nature of favorable osmolyte-protein interactions for improved thermal stability. This approach may be beneficial in the design of novel protein stabilizers with optimal property values, especially when the influence of solution conditions like the pH and buffer species and the protein properties are factored in.

Keywords: thermal stability, monoclonal antibodies, quantitative structure-activity relationships, osmolytes

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Authors: Narin Salehiyan, Ramin Ghasemi Shayan

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In order to investigate coronavirus RNA replication, transcription, recombination, protein processing and transport, virion assembly, the identification of coronavirus-specific cell receptors, and polymerase processing, the manipulation of coronavirus clones and complementary DNAs (cDNAs) of defective-interfering (DI) RNAs is the subject of this chapter. The idea of the Covid genome is nonsegmented, single-abandoned, and positive-sense RNA. When compared to other RNA viruses, its size is significantly greater, ranging from 27 to 32 kb. The quality encoding the enormous surface glycoprotein depends on 4.4 kb, encoding a forcing trimeric, profoundly glycosylated protein. This takes off exactly 20 nm over the virion envelope, giving the infection the appearance-with a little creative mind of a crown or coronet. Covid research has added to the comprehension of numerous parts of atomic science as a general rule, like the component of RNA union, translational control, and protein transport and handling. It stays a fortune equipped for creating startling experiences.

Keywords: covid-19, corona, virus, genome, genetic

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

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

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

Procedia PDF Downloads 276

Authors: Ayman El Behiry, Rasha Nabil Zahran, Reda Tarabees, Eman Marzouk, Musaad Al-Dubaib

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Mastitis is one of the most economic disease affecting dairy cows worldwide. Its classic diagnosis using bacterial culture and biochemical findings is a difficult and prolonged method. In this research, using of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) permitted identification of different microorganisms with high accuracy and rapidity (only 24 hours for microbial growth and analysis). During the application of MALDI-TOF MS, one hundred twenty strains of Staphylococcus and Streptococcus species isolated from milk of cows affected by clinical and subclinical mastitis were identified, and the results were compared with those obtained by traditional methods as API and VITEK 2 Systems. 37 of totality 39 strains (~95%) of Staphylococcus aureus (S. aureus) were exactly detected by MALDI TOF MS and then confirmed by a nuc-based PCR technique, whereas accurate identification was observed in 100% (50 isolates) of the coagulase negative staphylococci (CNS) and Streptococcus agalactiae (31 isolates). In brief, our results demonstrated that MALDI-TOF MS is a fast and truthful technique which has the capability to replace conventional identification of several bacterial strains usually isolated in clinical laboratories of microbiology.

Keywords: identification, mastitis pathogens, mass spectral, phenotypical

Procedia PDF Downloads 336

Authors: M. Kazou, A. Gavriil, O. Kalagkatsi, T. Paschos, E. Tsakalidou

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Kopanisti cheese is a Greek soft Protected Designation of Origin (PDO) cheese made of raw cow, sheep or goat milk, or mixtures of them, with similar organoleptic characteristics to that of Roquefort cheese. Traditional manufacturing of Kopanisti cheese is limited in small-scale dairies, without the addition of starter cultures. Instead, an amount of over-mature Kopanisti cheese, called Mana Kopanisti, is used to initiate ripening. Therefore, the selection of proper starter cultures and the understanding of the contribution of various microbial groups to its overall quality is crucial for the production of a high-quality final product with standardized organoleptic and physicochemical characteristics. Taking the above into account, the aim of the present study was the investigation of Kopanisti cheese microbiota and its role in cheese quality. For this purpose, four different types of Kopanisti were produced in triplicates, all with pasteurized cow milk, with the addition of (A) the typical mesophilic species Lactococcus lactis and Lactobacillus paracasei used as starters in the production of soft spread cheeses, (B) strains of Lactobacillus acidipiscis and Lactobacillus rennini previously isolated from Kopanisti and Mana Kopanisti, (C) all the species from (A) and (B) as inoculum, and finally (D) the species from (A) and Mana Kopanisti. Physicochemical and microbiological analysis was performed for milk and cheese samples during ripening. Enumeration was performed for major groups of lactic acid bacteria (LAB), total mesophilic bacteria, yeasts as well as hygiene indicator microorganisms. Bacterial isolates from all the different LAB groups, apart from enterococci, alongside yeasts isolates, were initially grouped using repetitive sequence-based polymerase chain reaction (rep-PCR) and then identified at the species level using 16S rRNA gene and internal transcribed spacer (ITS) DNA region sequencing, respectively. Sensory evaluation was also performed for final cheese samples at the end of the ripening period (35 days). Based on the results of the classical microbiological analysis, the average counts of the total mesophilic bacteria and LAB, apart from enterococci, ranged between 7 and 10 log colony forming unit (CFU) g⁻¹, phychrotrophic bacteria, and yeast extract glucose chloramphenicol (YGC) isolates between 4 and 8 log CFU g⁻¹, while coliforms and enterococci up to 2 log CFU g⁻¹ throughout ripening in cheese samples A, C and D. In contrast, in cheese sample B, the average counts of the total mesophilic bacteria and LAB, apart from enterococci, phychrotrophic bacteria, and YGC isolates ranged between 0 and 10 log CFU g⁻¹ and coliforms and enterococci up to 2 log CFU g⁻¹. Although the microbial counts were not that different among samples, identification of the bacterial and yeasts isolates revealed the complex microbial community structure present in each cheese sample. Differences in the physicochemical characteristics among the cheese samples were also observed, with pH ranging from 4.3 to 5.3 and moisture from 49.6 to 58.0 % in the final cheese products. Interestingly, the sensory evaluation also revealed differences among samples, with cheese sample B ranking first based on the total score. Overall, the combination of these analyses highlighted the impact of different starter cultures on the Kopanisti microbiota as well as on the physicochemical and sensory characteristics of the final product.

Keywords: Kopanisti cheese, microbiota, classical microbiological analysis, physicochemical analysis

Procedia PDF Downloads 135

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

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

Keywords: cannabidiol, resistance, Salmonella, antimicrobials, phages

Procedia PDF Downloads 71

Authors: Chuanpeng Zhou

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

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

Procedia PDF Downloads 459

Authors: Shagufta Jabeen, Faez J. Firdaus Abdullah, Zunita Zakaria, Nurulfiza M. Isa, Yung C. Tan, Wai Y. Yee, Abdul R. Omar

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Pasteurella multocida (PM) is an important veterinary opportunistic pathogen particularly associated with septicemic pasteurellosis, pneumonic pasteurellosis and hemorrhagic septicemia in cattle and buffaloes. P. multocida serotype A has been reported to cause fatal pneumonia and septicemia. Pasteurella multocida subspecies multocida of serotype A Malaysian isolate PMTB2.1 was first isolated from buffaloes died of septicemia. In this study, the genome of P. multocida strain PMTB2.1 was sequenced using third-generation sequencing technology, PacBio RS2 system and analyzed bioinformatically via de novo analysis followed by in-depth analysis based on comparative genomics. Bioinformatics analysis based on de novo assembly of PacBio raw reads generated 3 contigs followed by gap filling of aligned contigs with PCR sequencing, generated a single contiguous circular chromosome with a genomic size of 2,315,138 bp and a GC content of approximately 40.32% (Accession number CP007205). The PMTB2.1 genome comprised of 2,176 protein-coding sequences, 6 rRNA operons and 56 tRNA and 4 ncRNAs sequences. The comparative genome sequence analysis of PMTB2.1 with nine complete genomes which include Actinobacillus pleuropneumoniae, Haemophilus parasuis, Escherichia coli and five P. multocida complete genome sequences including, PM70, PM36950, PMHN06, PM3480, PMHB01 and PMTB2.1 was carried out based on OrthoMCL analysis and Venn diagram. The analysis showed that 282 CDs (13%) are unique to PMTB2.1and 1,125 CDs with orthologs in all. This reflects overall close relationship of these bacteria and supports the classification in the Gamma subdivision of the Proteobacteria. In addition, genomic distance analysis among all nine genomes indicated that PMTB2.1 is closely related with other five Pasteurella species with genomic distance less than 0.13. Synteny analysis shows subtle differences in genetic structures among different P.multocida indicating the dynamics of frequent gene transfer events among different P. multocida strains. However, PM3480 and PM70 exhibited exceptionally large structural variation since they were swine and chicken isolates. Furthermore, genomic structure of PMTB2.1 is more resembling that of PM36950 with a genomic size difference of approximately 34,380 kb (smaller than PM36950) and strain-specific Integrative and Conjugative Elements (ICE) which was found only in PM36950 is absent in PMTB2.1. Meanwhile, two intact prophages sequences of approximately 62 kb were found to be present only in PMTB2.1. One of phage is similar to transposable phage SfMu. The phylogenomic tree was constructed and rooted with E. coli, A. pleuropneumoniae and H. parasuis based on OrthoMCL analysis. The genomes of P. multocida strain PMTB2.1 were clustered with bovine isolates of P. multocida strain PM36950 and PMHB01 and were separated from avian isolate PM70 and swine isolates PM3480 and PMHN06 and are distant from Actinobacillus and Haemophilus. Previous studies based on Single Nucleotide Polymorphism (SNPs) and Multilocus Sequence Typing (MLST) unable to show a clear phylogenetic relatedness between Pasteurella multocida and the different host. In conclusion, this study has provided insight on the genomic structure of PMTB2.1 in terms of potential genes that can function as virulence factors for future study in elucidating the mechanisms behind the ability of the bacteria in causing diseases in susceptible animals.

Keywords: comparative genomics, DNA sequencing, phage, phylogenomics

Procedia PDF Downloads 188

Authors: A. Ameur Ameur, F. Chougrani, M. Halbouche

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In order to characterize the sheep's milk, we analyzed and compared, in a first stage of our work, the physical and chemical characteristics in two Algerian sheep breeds: Hamra race and race Ouled Djellal breeding at the station the experimental ITELV Ain Hadjar (Saïda Province). Analyses are performed by Ekomilk Ultra-analyzer (EON TRADING LLC, USA), they focused on the pH, density, freezing, fat, total protein, solids-the total dry extract. The results obtained for these parameters showed no significant differences between the two breeds studied. The second stage of this work was the isolation and characterization of milk proteins. For this, we used the precipitation of caseins phi [pH 4.6]. For this, we used the precipitation of caseins Phi (pH 4.6). After extraction, purification and assay, both casein and serum protein fractions were then assayed by the Bradford method and controlled by polyacrylamide gel electrophoresis (PAGE) in the different conditions (native, in the presence of urea and in the presence of SDS). The electrophoretic pattern of milk samples showed the presence similarities of four major caseins variants (αs1-, αs2-β-and k-casein) and two whey proteins (β-lactoglobulin, α-lactalbumin) of two races Hamra and Ouled Djellal. But compared to bovine milk, they have helped to highlight some peculiarities as related to serum proteins (α La β Lg) as caseins, including αs1-Cn.

Keywords: Hamra, Ouled Djellal, protein polymorphism, sheep breeds

Procedia PDF Downloads 558

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

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

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

Procedia PDF Downloads 388

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

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

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

Procedia PDF Downloads 69