Search results for: protein fortification

Authors: Charalampos N. Bompas, Eleni Poulou-Sidiropoulou, Martina Samiotaki, Alexios Vlamis-Gardikas

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Ιn all living organisms, antioxidant defenses are orchestrated by the thioredoxin (Trx) and glutaredoxin (Grx) systems. The Trx system of Escherichia coli (E. coli) is comprised of Trx1 and Trx2, both reduced by thioredoxin reductase (TrxR). The Grx system consists of four Grxs (Grx1, Grx2, Grx3, and Grx4), all reduced by glutathione (GSH) except for Grx4, which is reduced by TrxR. Under normal conditions, the GSH reductase of the Grx system keeps GSH at its reduced state. NADPH+ provides the electrons for all reductions in the Trx and Grx systems. Although the role of the E. coli Trx system is widely known, the function of the Grx system reflects the main property of Grx1, which is the reduction of ribonucleotide reductase Ia (RRIa). E. coli Grx3 (encoded by grxC) may also reduce RRIa in vitro but with slow kinetics. The molecule may account for up to 0.4% of total soluble protein and has been the subject of extensive structural studies. Its biological function, however, remains unknown. Herein, affinity chromatography with monothiol Grx3 serving as bait was used to detect the interactions of Grx3 with other proteins. Different types of interactions were identified (covalent, weak, and strong non-covalent) that suggested novel functions for Grx3. In silico approaches were employed to validate selected interactions. In addition, total protein extracts from the null mutant for grxC and the wild-type strain were compared. The overall findings suggest that Grx3 is involved in various metabolic processes, protein synthesis, and stress responses, expanding the recognized functions of Grx3 beyond the possible reduction of RRIa.

Keywords: escherichia coli, glutaredoxin 3, interactome, thiol-disulfide oxidoreductase

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Authors: Lydia Tan, Philippe Lemey, Lieselot Houspie, Marco Viveen, Darren Martin, Frank Coenjaerts

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Introduction: Human respiratory syncytial virus (hRSV) is the leading cause of severe respiratory tract infections in infants under the age of two. Genomic substitutions and related evolutionary dynamics of hRSV are of great influence on virus transmission behavior. The evolutionary patterns formed are due to a precarious interplay between the host immune response and RSV, thereby selecting the most viable and less immunogenic strains. Studying genomic profiles can teach us which genes and consequent proteins play an important role in RSV survival and transmission dynamics. Study design: In this study, genetic diversity and evolutionary rate analysis were conducted on 36 RSV subgroup B whole genome sequences and 37 subgroup A genome sequences. Clinical RSV isolates were obtained from nasopharyngeal aspirates and swabs of children between 2 weeks and 5 years old of age. These strains, collected during epidemic seasons from 2001 to 2011 in the Netherlands and Belgium by either conventional or 454-sequencing. Sequences were analyzed for genetic diversity, recombination events, synonymous/non-synonymous substitution ratios, epistasis, and translational consequences of mutations were mapped to known 3D protein structures. We used Bayesian statistical inference to estimate the rate of RSV genome evolution and the rate of variability across the genome. Results: The A and B profiles were described in detail and compared to each other. Overall, the majority of the whole RSV genome is highly conserved among all strains. The attachment protein G was the most variable protein and its gene had, similar to the non-coding regions in RSV, more elevated (two-fold) substitution rates than other genes. In addition, the G gene has been identified as the major target for diversifying selection. Overall, less gene and protein variability was found within RSV-B compared to RSV-A and most protein variation between the subgroups was found in the F, G, SH and M2-2 proteins. For the F protein mutations and correlated amino acid changes are largely located in the F2 ligand-binding domain. The small hydrophobic phosphoprotein and nucleoprotein are the most conserved proteins. The evolutionary rates were similar in both subgroups (A: 6.47E-04, B: 7.76E-04 substitution/site/yr), but estimates of the time to the most recent common ancestor were much lower for RSV-B (B: 19, A: 46.8 yrs), indicating that there is more turnover in this subgroup. Conclusion: This study provides a detailed description of whole RSV genome mutations, the effect on translation products and the first estimate of the RSV genome evolution tempo. The immunogenic G protein seems to require high substitution rates in order to select less immunogenic strains and other conserved proteins are most likely essential to preserve RSV viability. The resulting G gene variability makes its protein a less interesting target for RSV intervention methods. The more conserved RSV F protein with less antigenic epitope shedding is, therefore, more suitable for developing therapeutic strategies or vaccines.

Keywords: drug target selection, epidemiology, respiratory syncytial virus, RSV

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Authors: Tamar Khardziani, Violeta Berikashvili, Amrosi Chkuaseli, Vladimir Elisashvili

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The main objectives of this proposal are to develop low-cost, innovative, and competitive technologies for the production of mycelial biomass of medicinal mushrooms as a natural food supplement for poultry. To fulfill this task, industrial strains of Lentinus edodes, Ganoderma lucidum, and Pleurotus ostreatus were used in this study. The solid-state fermentation (SSF) of wheat grains, wheat bran, and soy flour was performed in flasks and bags. Among nine mushroom strains, P. ostreatus 2191 appeared to be the most productive in protein biomass accumulation in the SSF of wheat bran. All mushrooms produced exopolysaccharide with the highest yield of 5-8 mg/mL depending on fungal strain and growth substrate. Supplementation of medium with 1% glycerol and 2-4% peptone favored mushroom growth and protein accumulation. Among inorganic nitrogen sources, KNO₃ also provided high biomass and protein production. The SSF of all growth substrates was accompanied by the secretion of cellulase and xylanase activities. The highest CMCase activity (12-13 U/g) was revealed in the cultivation of P. ostreatus 2191 using wheat bran as a growth substrate and ammonium sulfate or yeast extract as a nitrogen source, whereas the highest xylanase activity was detected in the fermentation of soy flour supplemented with peptone. Acknowledgments: This work was supported by the Shota Rustaveli National Science Foundation of Georgia (Grant number STEM-22-2077).

Keywords: mushrooms, plant raw materials, fermentation, biomass protein, cellulase

Procedia PDF Downloads 43

Authors: Nihan Nugay, Nur Cicek Kekec, Kalman Toth, Turgut Nugay, Joseph P. Kennedy

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In recent years, polyurethane structures are paving the way for elastomer usage in biology, human medicine, and biomedical application areas. Polyurethanes having a combination of high oxidative and hydrolytic stability and excellent mechanical properties are focused due to enhancing the usage of PUs especially for implantable medical device application such as cardiac-assist. Currently, unique polyurethanes consisting of polyisobutylenes as soft segments and conventional hard segments, named as PIB-based PUs, are developed with precise NCO/OH stoichiometry (∽1.05) for obtaining PIB-based PUs with enhanced properties (i.e., tensile stress increased from ∽11 to ∽26 MPa and elongation from ∽350 to ∽500%). Static and dynamic mechanical properties were optimized by examining stress-strain graphs, self-organization and crystallinity (XRD) traces, rheological (DMA, creep) profiles and thermal (TGA, DSC) responses. Annealing procedure was applied for PIB-based PUs. Annealed PIB-based PU shows ∽26 MPa tensile strength, ∽500% elongation, and ∽77 Microshore hardness with excellent hydrolytic and oxidative stability. The surface characters of them were examined with AFM and contact angle measurements. Annealed PIB-based PU exhibits the higher segregation of individual segments and surface hydrophobicity thus annealing significantly enhances hydrolytic and oxidative stability by shielding carbamate bonds by inert PIB chains. According to improved surface and microstructure characters, greater efforts are focused on analyzing protein adsorption and calcification profiles. In biomedical applications especially for cardiological implantations, protein adsorption inclination on polymeric heart valves is undesirable hence protein adsorption from blood serum is followed by platelet adhesion and subsequent thrombus formation. The protein adsorption character of PIB-based PU examines by applying Bradford assay in fibrinogen and bovine serum albumin solutions. Like protein adsorption, calcium deposition on heart valves is very harmful because vascular calcification has been proposed activation of osteogenic mechanism in the vascular wall, loss of inhibitory factors, enhance bone turnover and irregularities in mineral metabolism. The calcium deposition on films are characterized by incubating samples in simulated body fluid solution and examining SEM images and XPS profiles. PIB-based PUs are significantly more resistant to hydrolytic-oxidative degradation, protein adsorption and calcium deposition than ElastEonTM E2A, a commercially available PDMS-based PU, widely used for biomedical applications.

Keywords: biomedical application, calcification, polyisobutylene, polyurethane, protein adsorption

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Authors: Md. Samsuddin Ansari, Ashish Arora

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Every year 10 million people fall ill with one of the oldest diseases known as tuberculosis, caused by Mycobacterium tuberculosis. The success of M. tuberculosis as a pathogen is because of its ability to persist in host tissues. Multidrug resistance (MDR) mycobacteria cases increase every day, which is associated with efflux pumps controlled at the level of transcription. The transcription regulators of MDR transporters in bacteria belong to one of the following four regulatory protein families: AraC, MarR, MerR, and TetR. Phenolic acid decarboxylase repressor (PadR), like a family of transcription regulators, is closely related to the MarR family. Phenolic acid decarboxylase repressor (PadR) was first identified as a transcription factor involved in the regulation of phenolic acid stress response in various microorganisms (including Mycobacterium tuberculosis H37Rv). Recently research has shown that the PadR family transcription factors are global, multifunction transcription regulators. Rv0047c is a PadR subfamily-1 protein. We are exploring the biophysical and structural characterization of Rv0047c. The Rv0047 gene was amplified by PCR using the primers containing EcoRI and HindIII restriction enzyme sites cloned in pET-NH6 vector and overexpressed in DH5α and BL21 (λDE3) cells of E. coli following purification with Ni2+-NTA column and size exclusion chromatography. We did DSC to know the thermal stability; the Tm (transition temperature) of protein is 55.29ºC, and ΔH (enthalpy change) of 6.92 kcal/mol. Circular dichroism to know the secondary structure and conformation and fluorescence spectroscopy for tertiary structure study of protein. To understand the effect of pH on the structure, function, and stability of Rv0047c we employed spectroscopy techniques such as circular dichroism, fluorescence, and absorbance measurements in a wide range of pH (from pH-2.0 to pH-12). At low and high pH, it shows drastic changes in the secondary and tertiary structure of the protein. EMSA studies showed the specific binding of Rv0047c with its own 30-bp promoter region. To determine the effect of complex formation on the secondary structure of Rv0047c, we examined the CD spectra of the complex of Rv0047c with promoter DNA of rv0047. The functional role of Rv0047c was characterized by over-expressing the Rv0047c gene under the control of hsp60 promoter in Mycobacterium tuberculosis H37Rv. We have predicted the three-dimensional structure of Rv0047c using the Swiss Model and Modeller, with validity checked by the Ramachandra plot. We did molecular docking of Rv0047c with dnaA, through PatchDock following refinement through FireDock. Through this, it is possible to easily identify the binding hot-stop of the receptor molecule with that of the ligand, the nature of the interface itself, and the conformational change undergone by the protein pattern. We are using X-crystallography to unravel the structure of Rv0047c. Overall the studies show that Rv0047c may have transcription regulation along with providing an insight into the activity of Rv0047c in the pH range of subcellular environment and helps to understand the protein-protein interaction, a novel target to kill dormant bacteria and potential strategy for tuberculosis control.

Keywords: mycobacterium tuberculosis, phenolic acid decarboxylase repressor, Rv0047c, Circular dichroism, fluorescence spectroscopy, docking, protein-protein interaction

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Authors: Kyung-Tae Lee, Li Li Wang, Kwang-Woo Jung, Yong-Sun Bahn

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Microtubules are involved in mechanical support, cytoplasmic organization as well as in a number of cellular processes by interacting with diverse microtubule-associated proteins (MAPs), such as plus-end tracking proteins, motor proteins, and tubulin-folding cofactors. A common feature of these proteins is the presence of a cytoskeleton-associated protein-glycine-rich (CAP-Gly) domain, which is evolutionarily conserved and generally considered to bind to α-tubulin to regulate functions of microtubules. However, there has been a dearth of research on CAP-Gly proteins in fungal pathogens, including Cryptococcus neoformans, which causes fatal meningoencephalitis globally. In this study, we identified five CAP-Gly proteins encoding genes in C. neoformans. Among these, Cgp1, encoded by CNAG_06352, has a unique domain structure that has not been reported before in other eukaryotes. Supporting the role of Cpg1 in microtubule-related functions, we demonstrate that deletion or overexpression of CGP1 alters cellular susceptibility to thiabendazole, a microtubule destabilizer, and Cgp1 is co-localized with cytoplasmic microtubules. Related to the cellular functions of microtubules, Cgp1 also governs maintenance of membrane stability and genotoxic stress responses. Furthermore, we demonstrate that Cgp1 uniquely regulates sexual differentiation of C. neoformans with distinct roles in the early and late stage of mating. Our domain analysis reveals that the CAP-Gly domain plays major roles in all the functions of Cgp1. Finally, the cgp1Δ mutant is attenuated in virulence. In conclusion, this novel CAP-Gly protein, Cgp1, has pleotropic roles in regulating growth, stress responses, differentiation and pathogenicity of C. neoformans.

Keywords: human fungal pathogen, CAP-Glycine protein, microtubule, meningoencephalitis

Procedia PDF Downloads 287

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 136

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 343

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 187

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 449

Authors: Zainal A. Muchlisin, Muhammad Iqbal, Abdullah A. Muhammadar

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The objective of the present study was to determine the optimum dose of papain enzyme in the diet for growing, survival rate and feed efficacy of climbing perch (Anabas testudineus). The study was conducted at the Laboratory of Aquatic of Faculty of Veterinary, Syiah Kuala University from January to March 2016. The completely randomized design was used in this study. Six dosages level of papain enzyme were tested with 4 replications i.e. 0 g kg-1 of feed, 20.0 g kg-1 feed, 22.5 g kg-1 of feed, 25.0 g kg-1 of feed, 27.5 g kg-1 of feed, and 30.0 g kg-1 of feed. The experimental fish fed twice a day at feeding level of 5% for 60 days. The results showed that weight gain ranged from 2.41g to 7.37g, total length gain ranged from 0.67cm to 3.17cm, specific growth rate ranged from 1.46 % day to 3.41% day, daily growth rate ranged from 0.04 g day to 0.13 g day, feed conversion ratio ranged from 1.94 to 3.59, feed efficiency ranged from 27.99% to 51.37%, protein retention ranged from 3.38% to 28.28%, protein digestibility ranged from 50.63% to 90.38%, and survival rate ranged from 88.89% to 100%. The highest rate for all parameters was found in the dosage of 3.00% papain enzyme kg feed. The ANOVA test showed that enzyme papain gave a significant effect on the weight gain, total length gain, daily growth rate, specific growth rate, feed conversion ratio, feed efficiency, protein retention, protein digestibility, and survival rate of the climbing perch (Anabas testudieus). The best enzyme papain dosage was 3.0%.

Keywords: betok, feed conversion ratio, freshwater fish, nutrition, feeding

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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

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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

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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

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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

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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

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

Abstract:

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

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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: Mohamed Yusuf

Abstract:

Aim: The growth promoting the effect of the blue-green filamentous alga Spirulina platensis (SP) was observed on meat type Japanese quail with antibiotic growth promoter alternative and immune enhancing power. Materials and Methods: This study was conducted on 180 Japanese quail chicks for 4 weeks to find out the effect of diet type (vegetarian protein diet [VPD] and fish meal protein diet [FMPD])- Spirulina dose interaction (1 or 2 g/kg diet) on growth performance, gut microbiota, and sensory meat quality of growing Japanese quails (1-5 weeks old). Results: Data revealed improvement (p<0.05) of weight gain, feed conversion ratio, and European efficiency index due to 1, 2 g (SP)/kg VPD, and 2 g (SP)/kg FMPD, respectively. There was a significant decrease of ileum mean pH value by 1 g(SP)/kg VPD. Concerning gut microbiota, there was a trend toward an increase in Lactobacilli count in both 1; 2 g (SP)/kgVPD and 2 g (SP)/kg FMPD. It was concluded that 1 or 2 g (SP)/kg vegetarian diet may enhance parameters of performance without obvious effect on both meat quality and gut microbiota. Moreover, 1 and/or 2 g (SP) may not be invited to share fishmeal based diet for growing Japanese quails. Conclusion: Using of SP will support the profitable production of Japanese quails fed vegetable protein diet.

Keywords: isocaloric, isonitrogenous, meat quality, performances, quails, spirulina, spirulina

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Authors: Khaled S. Al Salhen

Abstract:

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

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Authors: Ali Christian Chinedu, Asogwa Vincent Chidindu, Ejiofor Toochukwu Eleazar, Okadi Ashagwu Ojang

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The increasing rate of Boko Haram insurgency, farmer-herder clashes, and kidnapping in Nigeria has resulted in food shortages and high cost of protein sources like beef and fish. This challenge could be curbed with the production of edible insects, which contain several nutritional benefits like calories, protein, fat, vitamins, and minerals, depending on their species, metamorphic stage, and diet. Unfortunately, the benefits and competencies in producing, preserving, and marketing edible insects are still unknown to the public, including prospective farmers in Nigeria. Hence, this study determined teachers’ perception of integrating edible insects into the Animal Husbandry Curriculum in Senior Secondary Schools in Nigeria to equip the future generation with the relevant competencies for alternative sustainable protein supply. The study was carried out in Enugu State, Nigeria. The participants for the study comprised 162 agricultural science teachers. A questionnaire titled: Edible Insects Integration in Animal Husbandry Curriculum Questionnaire (EIIAHCQ) was used to collect data using a descriptive survey research design. We conducted data collection with the help of six research assistants. The study identified 11 objectives, 11 contents, 10 teaching methods, and 9 evaluation methods that could be integrated into the existing curriculum of animal husbandry in Nigeria. Among others, the Ministry of Education should integrate the finding of this study into the curriculum of Animal Husbandry in Nigeria to enhance the protein supply and curb food insecurity now and in the future.

Keywords: animal husbandry curriculum, edible insects, entomophagy, integration, secondary school, Nigeria

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

Abstract:

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

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Authors: Zhen Cao, Yu Zhu, Junxue Fu

Abstract:

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

Abstract:

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

Abstract:

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

Abstract:

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 246

Authors: K. Nazan Turhan, Tuğçe Ersan

Abstract:

The population of the world is approximately 7.6 billion, and it increases uncontrollably and irrepressibly, leading to an increase in consumption. This situation causes crucial problems, and food waste is one of these. Wasting food endangers natural resources and causes hunger. For instance, excessive amounts of food waste cause greenhouse gas emissions, contributing to global warming. Therefore, waste management has been gaining significance in the last few decades at both local and global levels due to the expected scarcity of resources for the increasing population of the world. There are several ways to recover food waste. Bioethanol, biodiesel, biogas, agricultural fertilizer and animal feed can be obtained from food waste that is generated by different food industries. In this project, feeding animals was selected as a food waste recovery method and food waste of a plant was used to provide ingredient uniformity. Grasshoppers were used as a protein source. In other words, the project was performed to develop a dog food product by recovering the plant’s food waste after following some steps. The collected food waste and purchased grasshoppers were sterilized, dried, and pulverized. Then, they were all mixed with 60 g agar-agar solution (4%w/v). 3 different aromas were added separately to the samples to enhance flavor quality. Since there are differences in the required amounts of different species of dogs, fulfilling all nutritional needs is one of the problems. In other words, there is a wide range of nutritional needs in terms of carbohydrates, protein, fat, sodium, calcium and so on. Furthermore, the requirements differ depending on age, gender, weight, height and species. Therefore, the product that was developed contains average amounts of each substance to not cause any deficiency or surplus. On the other hand, it contains more protein than similar products in the market. The product was evaluated in terms of contamination and nutritional content. For contamination risk, detection of E. coli and Salmonella experiments were performed, and the results were negative. For the nutritional value test, protein content analysis was done. The protein contents of different samples vary between 33.68% and 26.07%. In addition, water activity analysis was performed, and the water activity (aw) values of different samples ranged between 0.2456 and 0.4145.

Keywords: food waste, dog food, animal nutrition, food waste recovery

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