Search results for: fish sarcoplasmic protein

Authors: Maryam Ghamsari¹, Mitchell Nye-Wood², Kelvin Wang³, Angela Juhasz², Michelle Colgrave², Don Otter⁴, Jun Lu³, Nazimah Hamid¹, Thao T. Le¹

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

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

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Authors: Iftikhar A. Tayubi, Tabrej Khan, Mansoor M. Alsubei, Fahad A. Alsaferi

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LIZTOXD provides a single source of high-quality information about proteinaceous lizard toxins that will be an invaluable resource for pharmacologists, neuroscientists, toxicologists, medicinal chemists, ion channel scientists, clinicians, and structural biologists. We will provide an intuitive, well-organized and user-friendly web interface that allows users to explore the detail information of Lizard 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 Lizard, toxin and toxin protein of different Lizard species. These interfaces created in this database will satisfy the demands of the scientific community by providing in-depth knowledge about Lizard and its toxin. In the next phase of our project we will adopt methodology and by using A MySQL and Hypertext Preprocessor (PHP) which and for designing Smart Draw. A database is a wonderful piece of equipment for storing large quantities of data efficiently. 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, toxins, clinical data etc. LIZTOXD resource that provides comprehensive information about protein toxins from lizard toxins. The combination of specific classification schemes and a rich user interface allows researchers to easily locate and view information on the sequence, structure, and biological activity of these toxins. This manually curated database will be a valuable resource for both basic researchers as well as those interested in potential pharmaceutical and agricultural applications of lizard toxins.

Keywords: LIZTOXD, MySQL, PHP, smart draw

Procedia PDF Downloads 144

Authors: Prasanna Ramachandran, Kareem Graham, Helena Kiefel, Sunit Jain, Todd DeSantis

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Microbes that reside inside the mammalian GI tract, commonly referred to as the gut microbiome, have been shown to have therapeutic effects in animal models of disease. We hypothesize that specific proteins produced by these microbes are responsible for this activity and may be used directly as therapeutics. To speed up the discovery of these key proteins from the big-data metagenomics, we have applied machine learning techniques. Using amino acid sequences of known epitopes and their corresponding binding partners, protein interaction descriptors (PID) were calculated, making a positive interaction set. A negative interaction dataset was calculated using sequences of proteins known not to interact with these same binding partners. Using Random Forest and positive and negative PID, a machine learning model was trained and used to predict interacting versus non-interacting proteins. Furthermore, the continuous variable, cosine similarity in the interaction descriptors was used to rank bacterial therapeutic candidates. Laboratory binding assays were conducted to test the candidates for their potential as therapeutics. Results from binding assays reveal the accuracy of the machine learning prediction and are subsequently used to further improve the model.

Keywords: protein-interactions, machine-learning, metagenomics, microbiome

Procedia PDF Downloads 354

Authors: Sharon N. Nuñal, May Flor S. Muegue, Nizzy Hope N. Cartago, Raymund B. Parcon, Sheina B. Logronio

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The antioxidant and anti-inflammatory potentials of Perna Viridis, Modiolus philippinarum, and Mytella charruana found in the Philippines were assessed. Mussel protein samples were hydrolyzed using trypsin, maturase, alcalase and pepsin at 1% and 2% concentrations and then fractionated through membrane filtration (<10 kDa and <30 kDa). Antioxidant assays showed that pepsin hydrolysate at 2% enzyme concentration exhibited the maximum activities for both 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity (155-176 µM TE/mg protein) and 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging (67-68 µM TE/mg protein) assays while trypsin hydrolysate dominated the Ferric Reducing Antioxidant Power (FRAP) for the three mussel species. Lower molecular weight peptide fractions at <10 kDa exhibited better antioxidant activities than the higher molecular weight fractions. The anti-inflammatory activities of M. philippinarum and M. charruana showed comparable protein denaturation inhibition potentials with the highest in P. Viridis samples (98.93%). The 5-Lipoxygenase (5-LOX) inhibitory activities of mussel samples showed no significant difference with inhibition exceeding 70%. P. Viridis demonstrated the highest inhibition against Cyclooxygenase-2 (COX-2) at 56.19%, while the rest showed comparable activities. This study showed that the three mussel species are potential sources of bioactive peptides and lipids with antioxidant and anti-inflammatory properties.

Keywords: anti-inflammatory, antioxidant, bioactive properties, mussel

Procedia PDF Downloads 195

Authors: S. Reshma Reghu, Shiburaj Sugathan, T. G. Nandu, K. B. Ramesh Kumar, Mathew Dan

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Bacterial diseases are considered to be one of the most prevalent health hazards in the developing world and many bacteria are becoming resistant to existing antibiotics making the treatment ineffective. Thus, it is necessary to find novel targets and develop new antibacterial drugs with a novel mechanism of action. The process of bacterial cell division is a novel and attractive target for new antibacterial drug discovery. FtsZ, a homolog of eukaryotic tubulin, is the major protein of the bacterial cell division machinery and is considered as an important antibacterial drug target. Zingiberaceae, the Ginger family consists of aromatic herbs with creeping rhizomes. Many of these plants have antimicrobial properties.This study aimed to determine the anti-bacterial activity of selected Zingiberaceous plants by targeting bacterial cell division protein, FtsZ. Essential oils and methanol extracts of Amomum ghaticum, Alpinia galanga, Kaempferia galanga, K. rotunda, and Zingiber officinale were tested to find its antibacterial efficiency using disc diffusion method against authentic bacterial strains obtained from MTCC (India). Essential oil isolated from A.galanga and Z.officinale were further assayed for FtsZ inhibition assay following non-radioactive malachite green-phosphomolybdate assay using E. coli FtsZ protein obtained from Cytoskelton Inc., USA. Z.officinale essential oil possess FtsZ inhibitory property. A molecular docking study was conducted with the known bioactive compounds of Z. officinale as ligands with the E. coli FtsZ protein homology model. Some of the major constituents of this plant like catechin, epicatechin, and gingerol possess agreeable docking scores. The results of this study revealed that several chemical constituents in Ginger plants can be utilised as potential source of antibacterial activity and it can warrant further investigation through drug discovery studies.

Keywords: antibacterial, FtsZ, zingiberaceae, docking

Procedia PDF Downloads 457

Authors: Majid Javanmard

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In this study, the effects of coating with whey protein concentrate (7.5% w/v) alone and/or in combination with rice bran oil (0.2, 0.4, 0.6 g in 100 ml coating solution) and Zataria multiflora extract (1 and 2 μL in 100 ml coating solution) on the quality attributes and egg shelf life were carefully observed and analyzed. Weight loss, Haugh index, yolk index, pH, air cell depth, shell strength and the impact of this coating on the microbial load of the eggs surface were studied at the end of each week (during the 4 weeks of storage in a room environment temperature and humidity). After 4 weeks of storage, it was observed that the weight loss in all of the treated eggs with whey protein concentrate and 0.2 gr of rice bran oil (experimental group) was significantly lower than that of the control group(P < 0/05). With regard to Haugh index and yolk index, egg shelf life increased about 4 weeks compared with the control samples. Haugh Index changes revealed that the coated samples remained at grade A after 3 weeks of storage, while the control samples were relegated from grade AA to B after one week. Haugh and yolk Indices in all coated eggs were more than those of the control group. In the coated groups, Haugh and yolk indices of the coated samples with whey protein concentrate and 0.2 g rice bran oil and with whey protein concentrate and 0.2g of rice bran oil and 1 micro liter of Zataria multiflora extract were more than those of the other coated eggs and the control group eggs. PH values of the control group were higher than those of the coated groups during the storage of the eggs. The shell strength of the coated group was more than that of the control group (uncoated) and in coated samples, whey protein concentrate and 0.2 gr of rice bran oil coated samples had high shell strength. In the other treatments, no significant differences were observed. The depth of the air cell of the coated groups was determined to be less than that of the control group during the storage period. The minimum inhibitory concentration was 1 μL of Zataria multiflora extract. The results showed that 1 μL concentration of Zataria multiflora extract reduces the microbial load of the egg shell surface to 87% and 2 μL reduced total bacterial load to zero. In sensory evaluation, from evaluator point of view, the coated eggs had more overall acceptance than the uncoated group (control), and in the treatment group coated eggs, those containing a low percentage of rice bran oil had higher overall acceptability. In conclusion, coating as a practical and cost effective method can maintain the quality parameters of eggs and lead to durability of supply conditions in addition to the product marketability.

Keywords: edible coating, chicken egg, whey protein concentrate, rice bran oil, Zataria multiflora extract, shelf life

Procedia PDF Downloads 282

Authors: Ankita Singh, Chandan Gorain, Amirul I. Mallick

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Successful adherence of the mucosal epithelial cells is the key early step for Campylobacter jejuni pathogenesis (C. jejuni). A set of Surface Exposed Colonization Proteins (SECPs) are among the major factors involved in host cell adherence and invasion of C. jejuni. Among them, constitutively expressed surface-exposed lipoprotein adhesin of C. jejuni, JlpA, interacts with intestinal heat shock protein 90 (hsp90α) and contributes in disease progression by triggering pro-inflammatory response via activation of NF-κB and p38 MAP kinase pathway. Together with its ability to express in the bacterial surface, higher sequence conservation and predicted predominance of several B cells epitopes, JlpA protein reserves its potential to become an effective vaccine candidate against wide range of Campylobacter sps including C. jejuni. Given that chickens are the primary sources for C. jejuni and persistent gut colonization remain as major cause for foodborne pathogenesis to humans, present study explicitly used chickens as model to test the immune-protective efficacy of JlpA protein. Taking into account that gastrointestinal tract is the focal site for C. jejuni colonization, to extrapolate the benefit of mucosal (intragastric) delivery of JlpA protein, a food grade Nisin inducible Lactic acid producing bacteria, Lactococcus lactis (L. lactis) was engineered to express recombinant JlpA protein (rJlpA) in the surface of the bacteria. Following evaluation of optimal surface expression and functionality of recombinant JlpA protein expressed by recombinant L. lactis (rL. lactis), the immune-protective role of intragastric administration of live rL. lactis was assessed in commercial broiler chickens. In addition to the significant elevation of antigen specific mucosal immune responses in the intestine of chickens that received three doses of rL. lactis, marked upregulation of Toll-like receptor 2 (TLR2) gene expression in association with mixed pro-inflammatory responses (both Th1 and Th17 type) was observed. Furthermore, intragastric delivery of rJlpA expressed by rL. lactis, but not the injectable form, resulted in a significant reduction in C. jejuni colonization in chickens suggesting that mucosal delivery of live rL. lactis expressing JlpA serves as a promising vaccine platform to induce strong immune-protective responses against C. jejuni in chickens.

Keywords: chickens, lipoprotein adhesion of Campylobacter jejuni, immuno-protection, Lactococcus lactis, mucosal delivery

Procedia PDF Downloads 124

Authors: Aussara Panya, Nunghathai Sawasdee, Mutita Junking, Chatchawan Srisawat, Kiattawee Choowongkomon, Pa-Thai Yenchitsomanus

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Dengue virus (DENV) infection is a global public health problem with approximately 100 million infected cases a year. Presently, there is no approved vaccine or effective drug available; therefore, the development of anti-DENV drug is urgently needed. The clinical reports revealing the positive association between the disease severity and viral titer has been reported previously suggesting that the anti-DENV drug therapy can possibly ameliorate the disease severity. Although several anti-DENV agents showed inhibitory activities against DENV infection, to date none of them accomplishes clinical use in the patients. The surface envelope (E) protein of DENV is critical for the viral entry step, which includes attachment and membrane fusion; thus, the blocking of envelope protein is an attractive strategy for anti-DENV drug development. To search the safe anti-DENV agent, this study aimed to search for novel peptide inhibitors to counter DENV infection through the targeting of E protein using a structure-based in silico design. Two selected strategies has been used including to identify the peptide inhibitor which interfere the membrane fusion process whereby the hydrophobic pocket on the E protein was the target, the destabilization of virion structure organization through the disruption of the interaction between the envelope and membrane proteins, respectively. The molecular docking technique has been used in the first strategy to search for the peptide inhibitors that specifically bind to the hydrophobic pocket. The second strategy, the peptide inhibitor has been designed to mimic the ectodomain portion of membrane protein to disrupt the protein-protein interaction. The designed peptides were tested for the effects on cell viability to measure the toxic to peptide to the cells and their inhibitory assay to inhibit the DENV infection in Vero cells. Furthermore, their antiviral effects on viral replication, intracellular protein level and viral production have been observed by using the qPCR, cell-based flavivirus immunodetection and immunofluorescence assay. None of tested peptides showed the significant effect on cell viability. The small peptide inhibitors achieved from molecular docking, Glu-Phe (EF), effectively inhibited DENV infection in cell culture system. Its most potential effect was observed for DENV2 with a half maximal inhibition concentration (IC50) of 96 μM, but it partially inhibited other serotypes. Treatment of EF at 200 µM on infected cells also significantly reduced the viral genome and protein to 83.47% and 84.15%, respectively, corresponding to the reduction of infected cell numbers. An additional approach was carried out by using peptide mimicking membrane (M) protein, namely MLH40. Treatment of MLH40 caused the reduction of foci formation in four individual DENV serotype (DENV1-4) with IC50 of 24-31 μM. Further characterization suggested that the MLH40 specifically blocked viral attachment to host membrane, and treatment with 100 μM could diminish 80% of viral attachment. In summary, targeting the hydrophobic pocket and M-binding site on the E protein by using the peptide inhibitors could inhibit DENV infection. The results provide proof of-concept for the development of antiviral therapeutic peptide inhibitors to counter DENV infection through the use of a structure-based design targeting conserved viral protein.

Keywords: dengue virus, dengue virus infection, drug design, peptide inhibitor

Procedia PDF Downloads 340

Authors: E. Obreque-Slier, V. Contreras-Cortez, R. López-Solís

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Astringency is a drying, roughing, and sometimes puckering sensation that is experienced on the various oral surfaces during or immediately after tasting foods. This sensation has been closely related to the interaction and precipitation between salivary proteins and polyphenols, specifically flavanols or proanthocyanidins. In addition, the type and concentration of proanthocyanidin influences significantly the intensity of the astringency and consequently the protein/proanthocyanidin interaction. However, most of the studies are based on the interaction between saliva and highly complex polyphenols, without considering the effect of monomeric proanthoancyanidins present in different foods. The aim of this study was to evaluate the effect of different monomeric proanthocyanidins on the diffusion and precipitation of salivary proteins. Thus, solutions of catechin, epicatechin, epigallocatechin and gallocatechin (0, 2.0, 4.0, 6.0, 8.0 and 10 mg/mL) were mixed with human saliva (1: 1 v/v). After incubation for 5 min at room temperature, 15 µL aliquots of each mix were dotted on a cellulose membrane and allowed to dry spontaneously at room temperature. The membrane was fixed, rinsed and stained for proteins with Coomassie blue. After exhaustive washing in 7% acetic acid, the membrane was rinsed once in distilled water and dried under a heat lamp. Both diffusion area and stain intensity of the protein spots were semiqualitative estimates for protein-tannin interaction (diffusion test). The rest of the whole saliva-phenol solution mixtures of the diffusion assay were centrifuged, and 15-μL aliquots from each of the supernatants were dotted on a cellulose membrane. The membrane was processed for protein staining as indicated above. The blue-stained area of protein distribution corresponding to each of the extract dilution-saliva mixtures was quantified by Image J 1.45 software. Each of the assays was performed at least three times. Initially, salivary proteins display a biphasic distribution on cellulose membranes, that is, when aliquots of saliva are placed on absorbing cellulose membranes, and free diffusion of saliva is allowed to occur, a non-diffusible protein fraction becomes surrounded by highly diffusible salivary proteins. In effect, once diffusion has ended, a protein-binding dye shows an intense blue-stained roughly circular area close to the spotting site (non-diffusible fraction) (NDF) which becomes surrounded by a weaker blue-stained outer band (diffusible fraction) (DF). Likewise, the diffusion test showed that epicatechin caused the complete disappearance of DF from saliva with 2 mg/mL. Also, epigallocatechin and gallocatechin caused a similar effect with 4 mg/mL, while catechin generated the same effect at 8 mg/mL. In the precipitation test, the use of epicatechin and gallocatechin generated evident precipitates at the bottom of the Eppendorf tubes. In summary, the flavanol type differentially affects the diffusion and precipitation of saliva, which would affect the sensation of astringency perceived by consumers.

Keywords: astringency, polyphenols, tannins, tannin-protein interaction

Procedia PDF Downloads 185

Authors: Shahram Naghizadeh Raeisi, Ali Alghooneh, Seyed Jalal Razavi Zahedkolaei

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Herein, a shelf stable (no refrigeration required) UHT processed, aseptically packaged whey protein drink was formulated by using a new strategy in microparticulate process. Applying thermal and two-dimensional mechanical treatments simultaneously, a modified protein (MWPC-80) was produced. Then the physical, thermal and thermodynamic properties of MWPC-80 were assessed using particle size analysis, dynamic temperature sweep (DTS), and differential scanning calorimetric (DSC) tests. Finally, using MWPC-80, a new RTD beverage was formulated, and shelf stability was assessed for three months at ambient temperature (25 °C). Non-isothermal dynamic temperature sweep was performed, and the results were analyzed by a combination of classic rate equation, Arrhenius equation, and time-temperature relationship. Generally, results showed that temperature dependency of the modified sample was significantly (Pvalue<0.05) less than the control one contained WPC-80. The changes in elastic modulus of the MWPC did not show any critical point at all the processed stages, whereas, the control sample showed two critical points during heating (82.5 °C) and cooling (71.10 °C) stages. Thermal properties of samples (WPC-80 & MWPC-80) were assessed using DSC with 4 °C /min heating speed at 20-90 °C heating range. Results did not show any thermal peak in MWPC DSC curve, which suggested high thermal resistance. On the other hands, WPC-80 sample showed a significant thermal peak with thermodynamic properties of ∆G:942.52 Kj/mol ∆H:857.04 Kj/mole and ∆S:-1.22Kj/mole°K. Dynamic light scattering was performed and results showed 0.7 µm and 15 nm average particle size for MWPC-80 and WPC-80 samples, respectively. Moreover, particle size distribution of MWPC-80 and WPC-80 were Gaussian-Lutresian and normal, respectively. After verification of microparticulation process by DTS, PSD and DSC analyses, a 10% why protein beverage (10% w/w/ MWPC-80, 0.6% w/w vanilla flavoring agent, 0.1% masking flavor, 0.05% stevia natural sweetener and 0.25% citrate buffer) was formulated and UHT treatment was performed at 137 °C and 4 s. Shelf life study did not show any jellification or precipitation of MWPC-80 contained beverage during three months storage at ambient temperature, whereas, WPC-80 contained beverage showed significant precipitation and jellification after thermal processing, even at 3% w/w concentration. Consumer knowledge on nutritional advantages of whey protein increased the request for using this protein in different food systems especially RTD beverages. These results could make a huge difference in this industry.

Keywords: high protein whey beverage, micropartiqulation, two-dimentional mechanical treatments, thermodynamic properties

Procedia PDF Downloads 54

Authors: Sirilak Kongkaew, Pathumwadee Yodmanee, Nopporn Kaiyawet, Arthitaya Meeprasert, Thanyada Rungrotmongkol, Toshikatsu Kaburaki, Hiroshi Noguchi, Fujio Takeuch, Nawee Kungwan, Supot Hannongbua

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Behçet’s disease (BD) is a genetic autoimmune expressed by multisystemic inflammatory disorder mostly occurred at the skin, joints, gastrointestinal tract, and genitalia, including ocular, oral, genital, and central nervous systems. Most BD patients in Japan and Korea were strongly indicated by the genetic factor namely HLA-B*51 (especially, HLA-B*51:01) marker in HMC class I, while HLA-A*26:01 allele has been detected from the BD patients in Greek, Japan, and Taiwan. To understand the selective binding of the MICA-TM peptide towards the HLAs associated with BD, the molecular dynamics simulations were applied on the four HLA alleles (B*51:01, B*35:01, A*26:01, and A*11:01) in complex with such peptide. As a result, the key residues in the binding groove of HLA protein which play an important role in the MICA-TM peptide binding and stabilization were revealed. The Van der Waals force was found to be the main protein-protein interaction. Based on the binding free energy prediction by MM/PBSA method, the MICA-TM peptide interacted stronger to the HLA alleles associated to BD in the identical class by 7-12 kcal/mol. The obtained results from the present study could help to differentiate the HLA alleles and explain a source of Behçet’s disease.

Keywords: Behçet’s disease, MD simulations, HMC class I, autoimmune

Procedia PDF Downloads 383

Authors: Hani M. M. Alothaid, Louise Robson, Richmond Muimo

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This study will investigate cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) dependent calcineurin (Cn), known as protein phosphatase 2 B (PP2B) as well, regulation of chloride ion (Cl⁻) secretion and the release of pro-inflammatory molecules in immune cells such as cytokines. THP-1-derived monocytes, primary human monocytes and the bronchial epithelial cell line (16HBE14o-) were used in this study. The 16HBE14o- cells were chosen as positive control. Hence, to further confirm the expression of cystic fibrosis transmembrane conductance regulator (CFTR), calcium binding protein (S100A10), annexin A2 (AnxA2) and calcineurin A subunit (CnA) in all three cell types, cell lysate was probed against corresponding primary antibodies by immunoblotting. Western blot analyses show the expression of CFTR, AnxA2, CnA and S100A10 in THP-1-derived monocytes and primary human monocytes. In conclusion, CFTR, S100A10, CnA and AnxA2 are expressed in THP-1-derived monocytes and primary human monocytes and regulate Cl⁻ secretion. Also, they may play a role in the pro-inflammatory molecules release. The ongoing work will confirm interaction between these proteins in the cell lines.

Keywords: annexin A2, calcineurin, CFTR, chloride, monocytes, pro-inflammatory molecules, S100A10

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Authors: Khaled S. Al Salhen

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Aldehyde oxidase (AO) and xanthine oxidoreductase (XOR) catalyze the oxidation of many different N-heterocyclic compounds as well as aliphatic and aromatic aldehydes to their corresponding lactam and carboxylic acids respectively. The present study examines the oxidation of dimethylamino-cinnamaldehyde (DMAC), vanillin and phenanthridine by AO and xanthine by XOR from Drosophila cytosol. Therefore, the results obtained in the present study showed the DMAC, vanillin and phenanthridine substrates used were found to be good substrates of Drosophila AO and xanthine is the preferred substrate for Drosophila XOR. Km values of AO substrates were observed with DMAC (50±5.4 µM), phenanthridine (80±9.1 µM) and vanillin (303±11.7 µM) respectively for Drosophila cytosol. The Km values for DMAC and phenanthridine were ~6 and ~4 fold lower than that for vanillin as a substrate. The Km for XOR with xanthine using NAD+ as an electron acceptor was 27±4.1 µM. Relatively low Vmax values were obtained with phenanthridine (1.78±0.38 nmol/min/mg protein) and DMAC (1.80±0.35 nmol/min/mg protein). The highest Vmax was obtained from Drosophila cytosol with vanillin (7.58±2.11 nmol/min/mg protein). It is concluded these results that AO and XOR in Drosophila were able to catalyse the biotransformation of numerous substrates of the well-characterised mammalian AO and XOR. The kinetic parameters have indicated that the activity of AO of Drosophila may be a significant factor the oxidation of aromatic aldehyde compounds.

Keywords: aldehyde oxidase, xanthine oxidoreductase, dimethylamino-cinnamaldehyde, vanillin, phenanthridine, Drosophila melanogaster

Procedia PDF Downloads 428

Authors: Rahul Warmoota, Aditya Bhardwaj, Steffy Angural, Monika Rana, Sunena Jassal, Neena Puri, Naveen Gupta

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Seafood processing generates large volumes of waste products such as skin, heads, tails, shells, scales, backbones, etc. Pollution due to conventional methods of seafood waste disposal causes negative implications on the environment, aquatic life, and human health. Moreover, these waste products can be used for the production of high-value products which are still untapped due to inappropriate management. Paenibacillus sp. AD is known to act on chitinolytic and proteinaceous waste and was explored for its potential to degrade various types of seafood waste in solid-state fermentation. Effective degradation of seafood waste generated from a variety of sources such as fish scales, crab shells, prawn shells, and a mixture of such wastes was observed. 30 to 40 percent degradation in terms of decrease in the mass was achieved. Along with the degradation, chitinolytic and proteolytic enzymes were produced, which can have various biotechnological applications. Apart from this, value-added products such as chitin oligosaccharides and peptides of various degrees of polymerization were also produced, which can be used for various therapeutic purposes. Results indicated that Paenibacillus sp. AD can be used for the development of a process for the infield degradation of seafood waste.

Keywords: chitin, chitin-oligosaccharides, chitinase, protease, biodegradation, crab shells, prawn shells, fish scales

Procedia PDF Downloads 86

Authors: Mehmet Savsar

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Complex Life Support Systems (LSS) are used in all large commercial and public aquariums in order to keep the fish alive. Reliabilities of individual equipment, as well as the complete system, are extremely important and critical since the life and safety of important fish depend on these life support systems. Failure of some critical device or equipment, which do not have redundancy, results in negative consequences and affects life support as a whole. In this paper, we have considered a life support system in a large public aquarium in Kuwait Scientific Center and presented a procedure and analysis to show how the reliability of such systems can be estimated by using appropriate tools and collected data. We have also proposed possible improvements for systems reliability. In particular, addition of parallel components and spare parts are considered and the numbers of spare parts needed for each component to achieve a required reliability during specified lead time are calculated. The results show that significant improvements in system reliability can be achieved by operating some LSS components in parallel and having certain numbers of spares available in the spare parts inventories. The procedures and the results presented in this paper are expected to be useful for aquarium engineers and maintenance managers dealing with LSS.

Keywords: life support systems, aquariums, reliability, failures, availability, spare parts

Procedia PDF Downloads 268

Authors: Xian-Peng Jiang, Catherine C. Baucom, Toby Jiang, Robert L. Elliott

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HLA-G binds to the inhibitory receptors of uterine NK cells and plays an important role in protection of fetal cells from maternal NK lysis. HLA-G also mediates tumor escape, but the immunosuppressive role is often neglected. These studies have focused on the examination of HLA-G expression in human breast carcinoma and HLA-G immunosuppressive role in NK cytolysis. We examined HLA-G expression in breast cell lines by real time PCR, ELISA and immunofluorescent staining. We treated the breast cancer cell lines with anti-human HLA-G antibody or progesterone. Then, NK cytolysis was measured by using MTT assay. We find that breast carcinoma cell lines increase the expression of HLA-G mRNA and protein, compared to normal cells. Blocking HLA-G of the breast cancer cells by the antibody increases NK cytolysis. Progesterone upregulates HLA-G mRNA and protein of human breast cancer cell lines. The increased HLA-G expression suppresses NK cytolysis. In summary, human breast carcinoma overexpress HLA-G immunosuppressive molecules. Blocking HLA-G protein by antibody improves NK cytolysis. In contrast, upregulation of HLA-G expression by progesterone impairs NK cytolytic function. Thus, HLA-G is a new immunosuppressive checkpoint and potential cancer immunotherapeutic target.

Keywords: HLA-G, Breast carcinoma, NK cells, Immunosuppressive checkpoint

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Authors: Gajanan M. Sonwane

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The computational studies on 2-phenazinamines with their protein targets have been carried out to design compounds with potential anticancer activity. This strategy of designing compounds possessing selectivity over specific tyrosine kinase has been achieved through G-QSAR and molecular docking studies. The objective of this research has been to design newer 2-phenazinamine derivatives as Bcr-Abl tyrosine kinase inhibitors by G-QSAR, molecular docking studies followed by wet-lab studies along with evaluation of their anticancer potential. Computational chemistry was done by using VLife MDS 4.3 and Autodock 4.2 followed by wet-lab experiments for synthesizing 2-phenazinamine derivatives. The chemical structures of ligands in 2D were drawn by employing Chemdraw 2D Ultra 8.0 and were converted into 3D. These were optimized by using a semi-empirical method called MOPAC. The protein structure was retrieved from RCSC protein data bank as a PDB file. The binding interactions of protein and ligands were done by using PYMOL. The molecular properties of the designed compounds were predicted in silico by using Osiris property explorer. The parent compound 2-phenazinamine was synthesized by reduction of 2, 4-dinitro-N-phenyl-benzenamine in the presence of tin chloride followed by cyclization in the presence of nitrobenzene and magnesium sulfate. The derivatization at the amino function of 2-phenazinamine was performed by treating parent compound with various aldehydes in the presence of dicyclohexylcarbodiimide (DCC) and urea to afford 2-(2-chlorophenyl)-3-(phenazine-2-yl) thiazolidine-4-one. Synthesized 39 novel derivatives of 2-phenazinamine and performed antioxidant activity, anti antiproliferative on the bulb of onion and anticancer activity on cell line showing significant competition with marked blockbuster drug imatinib.

Keywords: computer-aided drug design, tyrosin kinases, anticancer, docking

Procedia PDF Downloads 124

Authors: Suhaila Qari, Samia Moharram, Safaa Alowaidi

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Objectives: To determine the fatty acids profiles in female fish, R. sarba from the Red Sea during the spawning season. Methods: Monthly individual Rhabdosargus sarba were obtained from Bangalah market in Jeddah, Red Sea and transported to the laboratory in ice aquarium. The total length, standard length and weight were measured, fishes were dissected. Ovaries were removed, weighed and 10 ml of concentrated hydrochloric acid were added to 10g of the ovary in a conical flask and immersed in boiling water until the sample was dissolved and the fat was seen to collect on the surface. The conical was cooled and the fat was extracted by shaking with 30 ml of diethyl ether. The extract was bowled after allowing the layers to separate into a weighed flask. The extraction was repeated three times more and distilled off the solvent then the fat dried at 100oC, cooled and weighed. Then 50 mg of lipid was put in a tube, 5 ml of methanolic sulphuric acid was added and 2 ml of benzene, the tube well closed and placed in water bath at 90oC for an hour and half. After cooling, 8 ml water and 5 ml petroleum was added shacked strongly and the ethereal layer was separated in a dry tube, evaporated to dryness. The fatty acid methyl esters were analyzed using a Hewlett Packard (HP 6890) chromatography, asplit /splitless injector and flame ionization detector (FID). Results: In female Rhabdosargus sarba, a total of 29 fatty acids detected in ovaries throughout the spawning season. The main fatty acid group in total lipid was saturated fatty acid (SFA, 28.9%), followed by 23.5% of polyunsaturated fatty acids (PUFA) and 12.9% of monounsaturated fatty acids (MUFA). The dominant SFA were palmitic and stearic, the major MUFA were palmitoleic and oleic, and the major PUFA were C18:2 and C22:2. During spawning stages no significant differences in total SFA, MUFA and PUFA, the highest value of SFA was in late spawning (36.78%). However, the highest value of MUFA and PUFA was in spawning (16.70% and 24.96% respectively). During spawning season there were a significant differences in total SFA between March (late spawning stage) and December (nearly ripe stage), (P < 0.05).

Keywords: sparidae, Rhabdosargus sarba, fish, fatty acids, spawning, gonads, red sea

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Authors: Punya Tepsing

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This research aimed to study the beliefs in auspicious materials of entrepreneurs in Muang Hat Yai. The data were collected via documentary research and field work including interviews, observations shops in Hat Yai which used auspicious materials to bring lucks to the shops. The results were as follows. The beliefs in auspicious materials that the entrepreneurs had were of three areas: 1) The auspicious materials could correct the improperness of the shop location, for example, the shop situated opposite a branch road, a shrine, or a bank. The owner usually corrected it by putting Chinese auspicious materials in front of or in the shop, for example, a lion holding a sword in his mouth, or a mirror, etc. 2) The auspicious materials could bring in more income. The owner of the shop usually put the auspicious materials such as a cat beckoning and a bamboo fish trap believed to trap money in front of or inside the shop. 3) The auspicious materials like turtles, paired fish and a monster holding the moon in his mouth could solve life problems including health, family, and safety problems. The use of these auspicious materials showed the blending of the beliefs of the Chinese shop entrepreneurs with the Thai folk beliefs. What is interesting is that Hat Yai is located near the three southern border provinces which are the unrest area and this may cause the number of tourists to decline. This prompted them to build a mechanism in adjusting themselves both to save their lives and to increase the number of customers. Auspicious materials can make them feel more confident.

Keywords: belief, auspicious materials, shop, entrepreneur, Maung Hat Yai

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Authors: Zhen Cao, Yu Zhu, Junxue Fu

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Immunoassay, i.e., antigen-antibody reaction, is crucial for disease diagnostics. To achieve the adequate signal of the antigen protein detection, a large amount of sample and long incubation time is needed. However, the amount of protein is usually small at the early stage, which makes it difficult to detect. Unlike cells and DNAs, no valid chemical method exists for protein amplification. Thus, an alternative way to improve the signal is through particle manipulation techniques to concentrate proteins, among which dielectrophoresis (DEP) is an effective one. DEP is a technique that concentrates particles to the designated region through a force created by the gradient in a non-uniform electric field. Since DEP force is proportional to the cube of particle size and square of electric field gradient, it is relatively easy to capture larger particles such as cells. For smaller ones like proteins, a super high gradient is then required. In this work, three-dimensional Ag/SiO2 nanorods arrays, fabricated by an easy physical vapor deposition technique called as oblique angle deposition, have been integrated with a DEP device and created the field gradient as high as of 2.6×10²⁴ V²/m³. The nanorods based DEP device is able to enrich bovine serum albumin (BSA) protein by 1800-fold and the rate has reached 180-fold/s when only applying 5 V electric potential. Based on the above nanorods integrated DEP platform, an immunoassay of mouse immunoglobulin G (IgG) proteins has been performed. Briefly, specific antibodies are immobilized onto nanorods, then IgG proteins are concentrated and captured, and finally, the signal from fluorescence-labelled antibodies are detected. The limit of detection (LoD) is measured as 275.3 fg/mL (~1.8 fM), which is a 20,000-fold enhancement compared with identical assays performed on blank glass plates. Further, prostate-specific antigen (PSA), which is a cancer biomarker for diagnosis of prostate cancer after radical prostatectomy, is also quantified with a LoD as low as 2.6 pg/mL. The time to signal saturation has been significantly reduced to one minute. In summary, together with an easy nanorod fabrication and integration method, this nanorods based DEP platform has demonstrated highly sensitive immunoassay performance and thus poses great potentials in applications for early point-of-care diagnostics.

Keywords: dielectrophoresis, immunoassay, oblique angle deposition, protein concentration

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Authors: Hadriana Bansi, Elizabeth Wina, Toto Toharmat

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Acacia villosa is thornless shrub legume which contents high crude protein. However, the utilization of A. villosa as ruminant feed is limited by its secondary compounds. The aim of this article is to find out the maximum of substitution A. villosa in sheep ration. The nutritional evaluation consisted of in vitro two stages, in vivo, and in vitro gas production trials. The secondary compounds of A. villosa also were analyzed. Evaluating digestibility of increasing level of substitution A. villosa replacing Pennisetum purpureum was using in vitro two stages. The substitution of 30% A. villosa was compared to 100% P. purpureum by in vitro gas production technique and in vivo digestibility. The results of two stages in vitro showed that total phenol, condensed tannin, and non-protein amino acid (NPAA) were high. Substitution 15% A. villosa reached the highest digestibility for both dry matter (DM) and crude protein (CP) which were 67% and 86% respectively, but it was shown that DM and CP digestibility of substitution 30% of A. villosa was still high which were 61.82% and 75-67% respectively. The pattern of gas production showed that first 8 hours total gas production substitution of 30% A. villosa was higher than 100% P. purpureum and declined after 10 hours incubation. In vivo trials showed that substitution of 30% A. villosa significantly increased CP intake, CP digestibility, and nitrogen retention. It can be concluded that substitution A. villosa until 30% still gave the good impact even though it has high secondary compounds.

Keywords: Acacia villosa, digestibility, gas production, secondary compounds

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Authors: Rahamdad Khan, Ijaz Ahmad Khan

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In modern agriculture the herbicides application are considered the most effective and fast in action against all types of weeds. But it’s a fact that the herbicide applicator cannot totally secure the crop plants from the possible herbicide injuries that further leads to several destructive changes in plant biochemistry. For the purpose pots studies were undertaken to test the tolerance order of chickpea against pre- emergence herbicides (Stomp 330 EC- Dual Gold 960 EC) and post- emergence herbicides (Topik 15 WP- Puma Super 75 EW- Isoproturon 500 EW) during 2012-13 and 2013-14. The experimental design was CRD with three replications. Plant height, number of branches plant-1, number of seeds plant-1, nodulation, seed protein contents and other growth related parameters in chickpea were examined during the investigations. The results indicate that all the enquire herbicides gave a significant variation to all recorded parameter of chick pea except nodule fresh and dray weight. Moreover the toxic effect of pre-emergence herbicide on chickpea was found higher as compared to post-emergence herbicides. Minimum chickpea plant height (50.50 cm), number of nodule plant-1 (17.83) and lowest seed protein (14.13 %) was recorded in Stomp 330 EC. Similarly the outmost seeds plant-1 (29.66) and number of nodule plant-1 (21) were found for Puma Super 75 EW. The results further showed that the highest seed protein content (21.75 and 21.15 %) was recorded for control/ untreated and Puma Super 75EW. Taking under concentration the possible negative impact of the herbicides the chemical application must be minimized up to certain extent at which the crop is mostly secure. However chemical weed control has many advantages so we should train our farmer regarding the proper use of agro chemical to minimize the loses in crops while using herbicides.

Keywords: chickpea, herbicides, protein, stomp 330 EC, weed

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Authors: Mehrnaz Mostafavi

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Recent advances in single-molecule protein identification (ID) and quantification techniques are poised to revolutionize proteomics, enabling researchers to delve into single-cell proteomics and identify low-abundance proteins crucial for biomedical and clinical research. This paper introduces a different approach to single-molecule protein ID and quantification using tri-color amino acid tags and a plasmonic nanopore device. A comprehensive simulator incorporating various physical phenomena was designed to predict and model the device's behavior under diverse experimental conditions, providing insights into its feasibility and limitations. The study employs a whole-proteome single-molecule identification algorithm based on convolutional neural networks, achieving high accuracies (>90%), particularly in challenging conditions (95–97%). To address potential challenges in clinical samples, where post-translational modifications affecting labeling efficiency, the paper evaluates protein identification accuracy under partial labeling conditions. Solid-state nanopores, capable of processing tens of individual proteins per second, are explored as a platform for this method. Unlike techniques relying solely on ion-current measurements, this approach enables parallel readout using high-density nanopore arrays and multi-pixel single-photon sensors. Convolutional neural networks contribute to the method's versatility and robustness, simplifying calibration procedures and potentially allowing protein ID based on partial reads. The study also discusses the efficacy of the approach in real experimental conditions, resolving functionally similar proteins. The theoretical analysis, protein labeler program, finite difference time domain calculation of plasmonic fields, and simulation of nanopore-based optical sensing are detailed in the methods section. The study anticipates further exploration of temporal distributions of protein translocation dwell-times and the impact on convolutional neural network identification accuracy. Overall, the research presents a promising avenue for advancing single-molecule protein identification and quantification with broad applications in proteomics research. The contributions made in methodology, accuracy, robustness, and technological exploration collectively position this work at the forefront of transformative developments in the field.

Keywords: nano proteomics, nanopore-based optical sensing, deep learning, artificial intelligence

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Authors: Ei Ei Hlaing, Narissara Lailerd, Sittiruk Roytrakul, Pichapat Piamrojanaphat

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Type 2 diabetes is one of the most common metabolic diseases all over the world. The pathogenesis of type 2 diabetes is not the only dysfunction of pancreatic beta cells but also insulin resistance in muscle, liver and adipose tissue. High levels of circulating free fatty acids, an increased lipid content of muscle cells, impaired insulin-mediated glucose uptake and diminished mitochondrial functioning are pathophysiological hallmarks of diabetic skeletal muscles. Purple rice (Oryza sativa L. indica) has been shown to have antidiabetic effects. However, the underlying mechanism(s) of antidiabetic activity of purple rice is still unraveled. In this research, to explore in-depth cellular mechanism(s), proteomic profile of purple rice supplemented type 2 diabetic rats’ skeletal muscle were analyzed contract with non-supplemented rats. Diabetic rats were induced high-fat diet combined with streptozotocin injection. By using one- dimensional gel electrophoresis (1-DE) and LC-MS/MS quantitative proteomic method, we analyzed proteomic profiles in skeletal muscle of normal rats, normal rats with purple rice supplementation, type 2 diabetic rats, and type 2 diabetic rats with purple rice supplementation. Total 2676 polypeptide expressions were identified. Among them, 24 peptides were only expressed in type 2 diabetic rats, and 24 peptides were unique peptides in type 2 diabetic rats with purple rice supplementation. Acetyl CoA carboxylase 1 (ACACA) found as unique protein in type 2 diabetic rats which is the major enzyme in lipid synthesis and metabolism. Interestingly, DNA damage response protein, heterogeneous nuclear ribonucleoprotein K [Mus musculus] (Hnrnpk), was upregulated in type 2 diabetic rats’ skeletal muscle. Meanwhile, unique proteins of type 2 diabetic rats with purple rice supplementation (bone morphogenetic 7 protein preproprotein, BMP7; and forkhead box protein NX4, Foxn4) involved with muscle cells growth through the regulation of TGF-β/Smad signaling network. Moreover, BMP7 may effect on insulin signaling through the downstream signaling of protein kinase B (Akt) which acts in protein synthesis, glucose uptake, and glycogen synthesis. In conclusion, our study supports that type 2 diabetes impairs muscular lipid metabolism. In addition, purple rice might recover the muscle cells growth and insulin signaling.

Keywords: proteomics, purple rice bran, skeletal muscle, type 2 diabetic rats

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Authors: P. N. Okeke, J. N. Chikwendu

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The study evaluated the effects of varying fermentation periods on the nutrients and anti-nutrients composition of African yam bean (Sphenostylis stenocarpa) and acha (Digitaria exilis) flour blends and sensory properties of their products. The African yam bean seeds and acha grains were fermented for 24 hrs, 48 and 72 hrs, dried (sun drying) and milled into fine flour. The fermented flours were used in a ratio of 70:30 (Protein basis) to formulate composite flour for meat pie and biscuits production. Both the fermented and unfermented flours and products were analyzed for chemical composition using the standard method. The data were statistically analyzed using SPSS version 15 to determine the mean and standard deviation. The 24, 48, and 72 hrs fermentation periods increased protein (22.81, 26.15 and 24.00% respectively). The carbohydrate, ash and moisture contents of the flours were also increased as a result of fermentation (68.01-76.83, 2.26-4.88, and 8.36-13.00% respectively). The 48 hrs fermented flour blends had the highest increase in ash relative to the control (4.88%). Fermentation increased zinc, iron, magnesium and phosphorus content of the flours. Treatment drastically reduced the anti-nutrient (oxalate, saponin, tannin, phytate, and hemagglutinin) levels of the flours. Both meat pie and biscuits had increased protein relative to the control (27.36-34.28% and 23.66-25.09%). However, the protein content of the meat pie increased more than that of the biscuits. Zinc, Iron, Magnesium and phosphorus levels increased in both meat pie and biscuits. Organoleptic attributes of the products (meat pie and biscuits) were slightly lower than the control except those of the 72 hrs fermented flours.

Keywords: fermentation, African yam bean, acha, biscuits, meat-pie

Procedia PDF Downloads 259

Authors: Özlem Ateş Sönmezoğlu, Begüm Terzi, Ahmet Yıldırım, Leyla Gündüz

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Durum wheat quality is defined as its suitability for pasta processing, that is pasta making quality. Another factor that determines the quality of durum wheat is the nutritional value of wheat or its final products. Wheat is a basic source of calories, proteins and minerals for humans in many countries of the world. For this reason, improvement of wheat nutritional value is of great importance. In recent years, deficiencies in protein and micronutrients, particularly in iron and zinc, have seriously increased. Therefore, basic foods such as wheat must be improved for micronutrient content. The effects of some major genes for grain quality established. Gpc-B1 locus is one of the genes increased protein and micronutrients content, and used in improvement studies of durum wheat nutritional value. The aim of this study was to increase the protein content and the micronutrient (Fe, Zn ve Mn) contents of an advanced durum wheat line (TMB 1) that was previously improved for its protein quality. For this purpose, TMB1 advanced durum wheat line were used as the recurrent parent and also, UC1113-Gpc-B1 line containing the Gpc-B1 gene was used as the gene source. In all of the generations, backcrossed plants carrying the targeted gene region were selected by marker assisted selection (MAS). BC4F1 plants MAS method was employed in combination with embryo culture and rapid plant growth in a controlled greenhouse conditions in order to shorten the duration of the transition between generations in backcross breeding. The Gpc-B1 gene was selected specific molecular markers. Since Yr-36 gene associated with Gpc-B1 allele, it was also transferred to the Gpc-B1 transferred lines. Thus, the backcrossed plants selected by MAS are resistance to yellow rust disease. This research has been financially supported by TÜBİTAK (112T910).

Keywords: Durum wheat, Gpc-B1, MAS, Triticum durum, Yr-36

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Authors: Orkide Donma, Mustafa M. Donma

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Obesity is associated with increased fat mass as well as fat percentage. Minerals are the elements, which are of vital importance. In this study, the relationships between body as well as bone mineral profile and the percentage as well as mass values of fat, fat-free portion, protein, skeletal muscle were evaluated in adult men with normal body mass index (N-BMI), and those classified according to different stages of obesity. A total of 103 adult men classified into five groups participated in this study. Ages were within 19-79 years range. Groups were N-BMI (Group 1), overweight (OW) (Group 2), first level of obesity (FLO) (Group 3), second level of obesity (SLO) (Group 4) and third level of obesity (TLO) (Group 5). Anthropometric measurements were performed. BMI values were calculated. Obesity degree, total body fat mass, fat percentage, basal metabolic rate (BMR), visceral adiposity, body mineral mass, body mineral percentage, bone mineral mass, bone mineral percentage, fat-free mass, fat-free percentage, protein mass, protein percentage, skeletal muscle mass and skeletal muscle percentage were determined by TANITA body composition monitor using bioelectrical impedance analysis technology. Statistical package (SPSS) for Windows Version 16.0 was used for statistical evaluations. The values below 0.05 were accepted as statistically significant. All the groups were matched based upon age (p > 0.05). BMI values were calculated as 22.6 ± 1.7 kg/m², 27.1 ± 1.4 kg/m², 32.0 ± 1.2 kg/m², 37.2 ± 1.8 kg/m², and 47.1 ± 6.1 kg/m² for groups 1, 2, 3, 4, and 5, respectively. Visceral adiposity and BMR values were also within an increasing trend. Percentage values of mineral, protein, fat-free portion and skeletal muscle masses were decreasing going from normal to TLO. Upon evaluation of the percentages of protein, fat-free portion and skeletal muscle, statistically significant differences were noted between NW and OW as well as OW and FLO (p < 0.05). However, such differences were not observed for body and bone mineral percentages. Correlation existed between visceral adiposity and BMI was stronger than that detected between visceral adiposity and obesity degree. Correlation between visceral adiposity and BMR was significant at the 0.05 level. Visceral adiposity was not correlated with body mineral mass but correlated with bone mineral mass whereas significant negative correlations were observed with percentages of these parameters (p < 0.001). BMR was not correlated with body mineral percentage whereas a negative correlation was found between BMR and bone mineral percentage (p < 0.01). It is interesting to note that mineral percentages of both body as well as bone are highly affected by the visceral adiposity. Bone mineral percentage was also associated with BMR. From these findings, it is plausible to state that minerals are highly associated with the critical stages of obesity as prominent parameters.

Keywords: bone, men, minerals, obesity

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Authors: Caspar S. Carson, Gabriel W. Prather, Nicholas E. Wong, Jeffery R. Anton, William H. McCoy

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Cutibacterium acnes is a commensal bacterium found on human skin that has been linked to acne. C. acnes can also be an opportunistic pathogen when it infiltrates the body during surgery. This pathogen can cause dangerous infections of medical implants, such as shoulder replacements, leading to life-threatening blood infections. Compounding this issue, C. acnes resistance to many antibiotics has become an increasing problem worldwide, creating a need for special forms of treatment. C. acnes expresses the protein RoxP, and it requires this protein to colonize human skin. Though this protein is required for C. acnes skin colonization, its function is not yet understood. Inhibition of RoxP function might be an effective treatment for C. acnes infections. To develop such reagents, the McCoy Laboratory generated four unique anti-RoxP antibodies. Preliminary studies in the McCoy Lab have established that each antibody binds a distinct site on RoxP. To assess the potential of these antibodies as therapeutics, it is necessary to specifically characterize these antibody epitopes and evaluate them in assays that assess their ability to inhibit RoxP-dependent C. acnes growth. To provide material for these studies, an antibody expression construct, Fv-clasp(v2), was adapted to encode anti-RoxP antibody sequences. The author hypothesizes that this expression strategy can produce sufficient amounts of >95% pure antibody fragments for further characterization of these antibodies. Four anti-RoxP Fv-clasp(v2) expression constructs (pET vector-based) were transformed into E. coli BL21-Gold(DE3) cells and a small-scale expression and purification trial was performed for each construct to evaluate anti-RoxP Fv-clasp(v2) yield and purity. Successful expression and purification of these antibody constructs will allow for their use in structural studies, such as protein crystallography and cryogenic electron microscopy. Such studies would help to define the antibody binding sites on RoxP, which could then be leveraged in the development of certain methods to treat C. acnes infection through RoxP inhibition.

Keywords: structural biology, protein expression, infectious disease, antibody, therapeutics, E. coli

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Authors: David Karasek, Veronika Kubickova, Ondrej Krystynik, Dominika Goldmannova, Lubica Cibickova, Jan Schovanek

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Introduction: Adiponectin, adipocyte-fatty acid-binding protein (A-FABP), and Wnt1 inducible signaling pathway protein-1 (WISP-1) are adipokines particularly associated with insulin resistance. The aim of the study was to compare their levels in women with gestational diabetes (GDM), type 2 diabetes mellitus (T2DM) and healthy controls and determine their relation with metabolic parameters. Methods: Fifty women with GDM, 50 women with T2DM, and 35 healthy women were included in the study. In addition to adipokines, anthropometric, lipid parameters, and markers, insulin resistance, and glucose control were assessed in all participants. Results: Compared to healthy controls only significantly lower levels of adiponectin were detected in women with GDM, whereas lower levels of adiponectin, higher levels of A-FABP and of WISP-1 were present in women with T2DM. Women with T2DM had also lower levels of adiponectin and higher levels of A-FABP compared to women with GDM. In women with GDM or T2DM adiponectin correlated negatively with body mass index (BMI), triglycerides (TG), C-peptide and positively with HDL-cholesterol; A-FABP positively correlated with BMI, TG, waist, and C-peptide. Moreover, there was a positive correlation between WISP-1 and C-peptide in women with T2DM. Conclusion: Adverse adipokines production detecting dysfunctional fat tissue is in women with GDM less presented than in women with T2DM, but more expressed compared to healthy women. Acknowledgment: Supported by AZV NV18-01-00139 and MH CZ DRO (FNOl, 00098892).

Keywords: adiponectin, adipocyte-fatty acid binding protein, wnt1 inducible signaling pathway protein-1, gestational diabetes, type 2 diabetes mellitus

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

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

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

Procedia PDF Downloads 87