Search results for: protein based

Authors: Aminah Salim, Idrees Ahmed Nasir, Abdul Qayyum Rao, Muhammad Ali, Muhammad Sohail Anjum, Ayesha Hameed, Bushra Tabassum, Anwar Khan, Arfan Ali, Mariyam Zameer, Tayyab Husnain

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Risk assessment of transgenic herbicide tolerant sugarcane having CEMB codon optimized cp4EPSPS gene was done in present study. Fifteen days old chicks taken from K&Ns Company were randomly assorted into four groups with eight chicks in each group namely control chicken group fed with commercial diet, non-transgenic group fed with non-experimental sugarcane and transgenic group fed with transgenic sugarcane with minimum and maximum level. Body weights, biochemical analysis for Urea, alkaline phosphatase, alanine transferase, aspartate transferase, creatinine and bilirubin determination and histological examination of chicks fed with four types of feed was taken at fifteen days interval and no significant difference was observed in body weight biochemical and histological studies of all four groups. Protein isolated from the serum sample was analyzed through dipstick and SDS-PAGE, showing the absence of transgene protein in the serum sample of control and experimental groups. Moreover the amplification of cp4EPSPS gene with gene specific primers of DNA isolated from chicks blood and also from commercial diet was done to determine the presence and mobility of any nucleotide fragment of the transgene in/from feed and no amplification was obtained in feed as well as in blood extracted DNA of any group. Also no mRNA expression of cp4EPSPS gene was obtained in any tissue of four groups of chicks. From the results it is clear that there is no deleterious or harmful effect of the CEMB codon optimized transgenic cp4EPSPS sugarcane on the chicks health.

Keywords: chicks, cp4EPSPS, glyphosate, sugarcane

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Authors: Fabienne Vozy, Anick Lepage

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Insects are displaying high nutritional value, low greenhouse gas emissions, low land use requirements and high food conversion efficiency. They can contribute to the food chain and be one of many solutions to protein shortages. Currently, in North America, nutritional entomology is under-developed and the needs to better understand its benefits remain to convince large-scale producers and consumers (both for human and agricultural needs). As such, large-scale production of mealworms offers a promising alternative to replacing traditional sources of protein and fatty acids. To proceed orderly, it is required to collect more data on the nutritional values of insects such as, a) Evaluate the diets of insects to improve their dietary value; b) Test the breeding conditions to optimize yields; c) Evaluate the use of by-products and organic residues as sources of food. Among the featured technical parameters, relative humidity (RH) percentage and temperature, optimal substrates and hydration sources are critical elements, thus establishing potential benchmarks for to optimize conversion rates of protein and fatty acids. This research is to establish the combination of the most influential rearing parameters with local food residues, to correlate the findings with the nutritional value of the larvae harvested. 125 same-monthly old adults/replica are randomly selected in the mealworm breeding pool then placed to oviposit in growth chambers preset at 26°C and 65% RH. Adults are removed after 7 days. Larvae are harvested upon the apparition of the first nymphosis signs and batches, are analyzed for their nutritional values using wet chemistry analysis. The first samples analyses include total weight of both fresh and dried larvae, residual humidity, crude proteins (CP%), and crude fats (CF%). Further analyses are scheduled to include soluble proteins and fatty acids. Although they are consistent with previous published data, the preliminary results show no significant differences between treatments for any type of analysis. Nutritional properties of each substrate combination have yet allowed to discriminate the most effective residue recipe. Technical issues such as the particles’ size of the various substrate combinations and larvae screen compatibility are to be investigated since it induced a variable percentage of lost larvae upon harvesting. To address those methodological issues are key to develop a standardized efficient procedure. The aim is to provide producers with easily reproducible conditions, without incurring additional excessive expenditure on their part in terms of equipment and workforce.

Keywords: entomophagy, nutritional value, rearing parameters optimization, Tenebrio molitor

Procedia PDF Downloads 111

Authors: Ayşe Defne Öz, Özlem Bozkurt

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Alzheimer's Disease (AD) is counted as one of the most important global health problems and the main cause of dementia. The term dementia refers to a wide spectrum of disorders characterized by global, chronic, and generally irreversible cognitive deterioration. It is estimated that %60 % to 80 of the cases of dementia are because of AD. Alzheimer's is a slowly progressive brain disease. The reason for AD is unknown to the author's best knowledge, yet it is one of the topics that is most researched. AD shows the histopathologically abnormal accumulation of the protein beta-amyloid (plague) outside neurons and twisted strands of the protein tau (tangles) inside neurons in the brain. These changes are accompanied by damage to the brain tissue and the death of neurons. AD causes people to have difficulty remembering names or conversations. Some of the later symptoms are difficulty in talking and walking. Alzheimer's Disease is elevated by the illness and mortality of COVID-19. COVID-19 has affected many lives globally and had profound effects on human lives. COVID-19 is caused by SARS-CoV-2, which is a virus that attacks the respiratory and central nervous system and has neuroinvasive potential. More than %80 of COVID-19 patients have ageusia or anosmia, representing the pathognomic features of the disease. Patients with dementia are frail, and with the COVID-19 pandemic, including isolation, cognitive decline may exacerbate. Furthermore, patients with AD can be unable to follow the directions, such as covering their mouth and nose while coughing and can live in nursing homes which makes them more open to being infected. As COVID-19 is highly infectious and its management requires isolation and quarantine, the need for caregivers for AD management conflicts with that of COVID-19 and adds an extra burden on AD patients, caregivers, families, society, and the economy. Due to the entry of SARS-CoV-2 into the central nervous system, inflammation caused by COVID-19, prolonged hospitalization, and delirium, it has been reported that COVID-19 causes many neurological disorders and predisposition to AD.

Keywords: Alzheimer's disease, COVID-19, dementia, SARS-CoV-2

Procedia PDF Downloads 76

Authors: Sanja O. Podunavac-Kuzmanović, Strahinja Z. Kovačević, Lidija R. Jevrić

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Prion is a protein substance whose certain form is considered as infectious agent. It is presumed to be the cause of the transmissible spongiform encephalopathies (TSEs). The protein it is composed of, called PrP, can fold in structurally distinct ways. At least one of those 3D structures is transmissible to other prion proteins. Prions can be found in brain tissue of healthy people and have certain biological role. The structure of prions naturally occurring in healthy organisms is marked as PrPc, and the structure of infectious prion is labeled as PrPSc. PrPc may play a role in synaptic plasticity and neuronal development. Also, it may be required for neuronal myelin sheath maintenance, including a role in iron uptake and iron homeostasis. PrPSc can be considered as an environmental pollutant. The main aim of this study was to carry out the molecular modeling and calculation of molecular descriptors (lipophilicity, physico-chemical and topological descriptors) of structurally diverse compounds which can be considered as anti-prion agents. Molecular modeling was conducted applying ChemBio3D Ultra version 12.0 software. The obtained 3D models were subjected to energy minimization using molecular mechanics force field method (MM2). The cutoff for structure optimization was set at a gradient of 0.1 kcal/Åmol. The Austin Model 1 (AM-1) was used for full geometry optimization of all structures. The obtained set of molecular descriptors is applied in analysis of similarities and dissimilarities among the tested compounds. This study is an important step in further development of quantitative structure-activity relationship (QSAR) models, which can be used for prediction of anti-prion activity of newly synthesized compounds.

Keywords: chemometrics, molecular modeling, molecular descriptors, prions, QSAR

Procedia PDF Downloads 322

Authors: Paula K. Novelli, Margarida M. Barros, Luciana F. Flueri

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Utilization of agricultural by-products, wheat ban and soybean bran, as substrate for solid state fermentation (SSF) was studied, aiming the achievement of different enzymes from Aspergillus sp. with distinct biological characteristics and its application and improvement on animal nutrition. Aspergillus niger and Aspergillus oryzea were studied as they showed very high yield of phytase and protease production, respectively. Phytase activity was measure using p-nitrophenilphosphate as substrate and a standard curve of p-nitrophenol, as the enzymatic activity unit was the quantity of enzyme necessary to release one μmol of p-nitrophenol. Protease activity was measure using azocasein as substrate. Activity for phytase and protease substantially increased when the different biochemical characteristics were considered in the study. Optimum pH and stability of the phytase produced by A. niger with wheat bran as substrate was between 4.0 - 5.0 and optimum temperature of activity was 37oC. Phytase fermented in soybean bran showed constant values at all pHs studied, for optimal and stability, but low production. Phytase with both substrates showed stable activity for temperatures higher than 80oC. Protease from A. niger showed very distinct behavior of optimum pH, acid for wheat bran and basic for soybean bran, respectively and optimal values of temperature and stability at 50oC. Phytase produced by A. oryzae in wheat bran had optimum pH and temperature of 9 and 37oC, respectively, but it was very unstable. On the other hand, proteases were stable at high temperatures, all pH’s studied and showed very high yield when fermented in wheat bran, however when it was fermented in soybean bran the production was very low. Subsequently the upscale production of phytase from A. niger and proteases from A. oryzae were applied as an enzyme additive in fish fed for digestibility studies. Phytases and proteases were produced with stable enzyme activity of 7,000 U.g-1 and 2,500 U.g-1, respectively. When those enzymes were applied in a plant protein based fish diet for digestibility studies, they increased protein, mineral, energy and lipids availability, showing that these new enzymes can improve animal production and performance. In conclusion, the substrate, as well as, the microorganism species can affect the biochemical character of the enzyme produced. Moreover, the production of these enzymes by SSF can be up to 90% cheaper than commercial ones produced with the same fungi species but submerged fermentation. Add to that these cheap enzymes can be easily applied as animal diet additives to improve production and performance.

Keywords: agricultural by-products, animal nutrition, enzymes production, solid state fermentation

Procedia PDF Downloads 326

Authors: Supria Sarkar, Vasit Sagan, Sourav Bhadra, Meghnath Pokharel, Felix B.Fritschi

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Soybean and their derivatives are very important agricultural commodities around the world because of their wide applicability in human food, animal feed, biofuel, and industries. However, the significance of soybean production depends on the quality of the soybean seeds rather than the yield alone. Seed composition is widely dependent on plant physiological properties, aerobic and anaerobic environmental conditions, nutrient content, and plant phenological characteristics, which can be captured by high temporal resolution remote sensing datasets. Planet scope (PS) satellite images have high potential in sequential information of crop growth due to their frequent revisit throughout the world. In this study, we estimate soybean seed composition while the plants are in the field by utilizing PlanetScope (PS) satellite images and different machine learning algorithms. Several experimental fields were established with varying genotypes and different seed compositions were measured from the samples as ground truth data. The PS images were processed to extract 462 hand-crafted vegetative and textural features. Four machine learning algorithms, i.e., partial least squares (PLSR), random forest (RFR), gradient boosting machine (GBM), support vector machine (SVM), and two recurrent neural network architectures, i.e., long short-term memory (LSTM) and gated recurrent unit (GRU) were used in this study to predict oil, protein, sucrose, ash, starch, and fiber of soybean seed samples. The GRU and LSTM architectures had two separate branches, one for vegetative features and the other for textures features, which were later concatenated together to predict seed composition. The results show that sucrose, ash, protein, and oil yielded comparable prediction results. Machine learning algorithms that best predicted the six seed composition traits differed. GRU worked well for oil (R-Squared: of 0.53) and protein (R-Squared: 0.36), whereas SVR and PLSR showed the best result for sucrose (R-Squared: 0.74) and ash (R-Squared: 0.60), respectively. Although, the RFR and GBM provided comparable performance, the models tended to extremely overfit. Among the features, vegetative features were found as the most important variables compared to texture features. It is suggested to utilize many vegetation indices for machine learning training and select the best ones by using feature selection methods. Overall, the study reveals the feasibility and efficiency of PS images and machine learning for plot-level seed composition estimation. However, special care should be given while designing the plot size in the experiments to avoid mixed pixel issues.

Keywords: agriculture, computer vision, data science, geospatial technology

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Authors: Leonard Lipovich, Pattaraporn Thepsuwan, Anton-Scott Goustin, Juan Cai, Donghong Ju, James B. Brown

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Two-thirds of human genes do not encode any known proteins. Aside from long non-coding RNA (lncRNA) genes with recently-discovered functions, the ~40,000 non-protein-coding human genes remain poorly understood, and a role for their transcripts as de-facto unconventional messenger RNAs has not been formally excluded. Ribosome profiling (Riboseq) predicts translational potential, but without independent evidence of proteins from lncRNA open reading frames (ORFs), ribosome binding of lncRNAs does not prove translation. Previously, we mass-spectrometrically documented translation of specific lncRNAs in human K562 and GM12878 cells. We now examined lncRNA translation in human MCF7 cells, integrating strand-specific Illumina RNAseq, Riboseq, and deep mass spectrometry in biological quadruplicates performed at two core facilities (BGI, China; City of Hope, USA). We excluded known-protein matches. UCSC Genome Browser-assisted manual annotation of imperfect (tryptic-digest-peptides)-to-(lncRNA-three-frame-translations) alignments revealed three peptides hypothetically explicable by 'stop-to-nonstop' in-frame replacement of stop codons by amino acids in two ORFs of the lncRNA MMP24-AS1. To search for this phenomenon genomewide, we designed and implemented a novel pipeline, matching tryptic-digest spectra to wildcard-instead-of-stop versions of repeat-masked, six-frame, whole-genome translations. Along with singleton putative stop-to-nonstop events affecting four other lncRNAs, we identified 24 additional peptides with stop-to-nonstop in-frame substitutions from multiple positive-strand MMP24-AS1 ORFs. Only UAG and UGA, never UAA, stop codons were impacted. All MMP24-AS1-matching spectra met the same significance thresholds as high-confidence known-protein signatures. Targeted resequencing of MMP24-AS1 genomic DNA and cDNA from the same samples did not reveal any mutations, polymorphisms, or sequencing-detectable RNA editing. This unprecedented apparent gene-specific violation of the genetic code highlights the importance of matching peptides to whole-genome, not known-genes-only, ORFs in mass-spectrometry workflows, and suggests a new mechanism enhancing the combinatorial complexity of the proteome. Funding: NIH Director’s New Innovator Award 1DP2-CA196375 to LL.

Keywords: genetic code, lncRNA, long non-coding RNA, mass spectrometry, proteogenomics, ribo-seq, ribosome, RNAseq

Procedia PDF Downloads 235

Authors: Yung-Feng Yen, I-an Jen, Yi-Ming Arthur Chen

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The molecular mimicry between human immunodeficiency virus (HIV) protein and red blood cell (RBC) antigens could induce the production of anti-RBC autoantibodies. However, the association between HIV infection and subsequent development of autoimmune hemolytic anemia (AIHA) remains unclear. This nationwide population-based cohort study aimed to determine the association between incident AIHA and HIV in Taiwan. From 2000–2012, we identified adult people living with HIV/AIDS (PLWHA) from the Taiwan centers for disease control HIV Surveillance System. HIV-infected individuals were defined by positive HIV-1 western blot. Age- and sex-matched controls without HIV infection were selected from the Taiwan National Health Insurance Research Database for comparison. All patients were followed until Dec. 31, 2012, and observed for occurrence of AIHA. Of 171,468 subjects (19,052 PLWHA, 152,416 controls), 30 (0.02%) had incident AIHA during a mean follow-up of 5.45 years, including 23 (0.12%) PLWHA and 7 (0.01%) controls. After adjusting for potential confounders, HIV infection was found to be an independent risk factor of incident AIHA (adjusted hazard ratio [AHR], 20.9; 95% confidence interval [CI], 8.34-52.3). Moreover, PLWHA receiving HAART were more likely to develop AIHA than those not receiving HAART (AHR, 10.8; 95% CI, 2.90-40.1). Additionally, the risk of AIHA was significantly increased in those taking efavirenz (AHR, 3.15; 95% CI, 1.18-8.43) or atazanavir (AHR, 6.58; 95% CI, 1.88-22.9) component of the HAART. In conclusion, HIV infection is an independent risk factor for incident AIHA. Clinicians need to be aware of the higher risk of AIHA in PLWHA.

Keywords: autoimmune disease , hemolytic anemia, HIV, highly active antiretroviral treatment

Procedia PDF Downloads 235

Authors: Elif Tugce Aksun Tumerkan

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Although seafood is an important part of human diet and categorized highly traded food industry internationally, it is remain overlooked generally in the global food security aspect. Food product authentication is the main interest in the aim of both avoids commercial fraud and to consider the risks that might be harmful to human health safety. In recent years, with increasing consumer demand for regarding food content and it's transparency, there are some instrumental analyses emerging for determining food fraud depend on some analytical methodologies such as proteomic and metabolomics. While, fish and seafood consumed as fresh previously, within advanced technology, processed or packaged seafood consumption have increased. After processing or packaging seafood, morphological identification is impossible when some of the external features have been removed. The main fish and seafood quality-related issues are the authentications of seafood contents such as mislabelling products which may be contaminated and replacement partly or completely, by lower quality or cheaper ones. For all mentioned reasons, truthful consistent and easily applicable analytical methods are needed for assurance the correct labelling and verifying of seafood products. DNA-barcoding methods become popular robust that used in taxonomic research for endangered or cryptic species in recent years; they are used for determining food traceability also. In this review, when comparing the other proteomic and metabolic analysis, DNA-based methods are allowing a chance to identification all type of food even as raw, spiced and processed products. This privilege caused by DNA is a comparatively stable molecule than protein and other molecules. Furthermore showing variations in sequence based on different species and founding in all organisms, make DNA-based analysis more preferable. This review was performed to clarify the main advantages of using DNA-barcoding for determining seafood fraud among other techniques.

Keywords: DNA-barcoding, genetic analysis, food fraud, mislabelling, packaged seafood

Procedia PDF Downloads 168

Authors: Pureun-Haneul Lee, Byeong-Gon Kim, Sun-Hye Lee, An-Soo Jang

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Background: Nanoparticles may pose adverse health effects due to particulate matter inhalation. Nanoparticle exposure induces cell and tissue damage, causing local and systemic inflammatory responses. The inflammasome is a major regulator of inflammation through its activation of pro-caspase-1, which cleaves pro-interleukin-1β (IL-1β) into its mature form and may signal acute and chronic immune responses to nanoparticles. Objective: The aim of the study was to identify whether nanoparticles exaggerates inflammasome pathway leading to airway inflammation and hyperresponsiveness in an allergic mice model of asthma. Methods: Mice were treated with saline (sham), OVA-sensitized and challenged (OVA), or titanium dioxide nanoparticles. Lung interleukin 1 beta (IL-1β), interleukin 18 (IL-18), NACHT, LRR and PYD domains-containing protein 3 (NLRP3) and caspase-1 levels were assessed with Western Blot. Caspase-1 was checked by immunohistochemical staining. Reactive oxygen species were measured for the marker 8-isoprostane and carbonyl by ELISA. Results: Airway inflammation and hyperresponsiveness increased in OVA-sensitized/challenged mice and these responses were exaggerated by TiO2 nanoparticles exposure. TiO2 nanoparticles treatment increased IL-1β and IL-18 protein expression in OVA-sensitized/challenged mice. TiO2 nanoparticles augmented the expression of NLRP3 and caspase-1 leading to the formation of an active caspase-1 in the lung. Lung caspase-1 expression was increased in OVA-sensitized/challenged mice and these responses were exaggerated by TiO2 nanoparticles exposure. Reactive oxygen species was increased in OVA-sensitized/challenged mice and in OVA-sensitized/challenged plus TiO2 exposed mice. Conclusion: Our data demonstrate that inflammasome pathway activates in asthmatic lungs following nanoparticles exposure, suggesting that targeting the inflammasome may help control nanoparticles-induced airway inflammation and responsiveness.

Keywords: bronchial asthma, inflammation, inflammasome, nanoparticles

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Authors: Uchenna Nkiruka Umeononihu, Adenike Kuku, Oludele Odekanyin, Olubunmi Babalola, Femi Agboola, Rapheal Okonji

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In this study, a lectin from the bulb of Dioscorea bulbifera was purified, characterised, and its acute and sub-acute toxicity was investigated with a view to evaluate its toxic effects in mice. The protein from the bulb was extracted by homogenising 50 g of the bulb in 500 ml of phosphate buffered saline (0.025 M) of pH 7.2, stirred for 3 hr, and centrifuged at the speed of 3000 rpm. Blood group and sugar specificity assays of the crude extract were determined. The lectin was purified in a two-step procedure- gel filtration on Sephadex G-75 and affinity chromatography on Sepharose 4-B arabinose. The degree of purity of the purified lectin was ascertained by SDS-polyacrylamide gel electrophoresis. Detection of covalently bound carbohydrate was carried out with Periodic Acid-Schiffs (PAS) reagent staining technique. Effects of temperature, pH, and EDTA on the lectin were carried out using standard methods. This was followed by acute toxicity studies via oral and subcutaneous routes using mice. The animals were monitored for mortality and signs of toxicity. The sub-acute toxicity studies were carried out using rats. Different concentrations of the lectin were administered twice daily for 5 days via the subcutaneous route. The animals were sacrificed on the sixth day; blood samples and liver tissues were collected. Biochemical assays (determination of total protein, direct bilirubin, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), catalase (CAT), and superoxide dismutase (SOD)) were carried out on the serum and liver homogenates. The collected organs (heart, liver, kidney, and spleen) were subjected to histopathological analysis. The results showed that lectin from the bulbs of Dioscorea bulbifera agglutinated non-specifically the erythrocytes of the human ABO system as well as rabbit erythrocytes. The haemagglutinating activity was strongly inhibited by arabinose and dulcitol with minimum inhibitory concentrations of 0.781 and 6.25, respectively. The lectin was purified to homogeneity with native and subunit molecular weights of 56,273 and 29,373 Daltons, respectively. The lectin was thermostable up to 30 0C and lost 25 %, 33.3 %, and 100 % of its heamagglutinating activity at 40°C, 50°C, and 60°C, respectively. The lectin was maximally active at pH 4 and 5 but lost its total activity at pH eight, while EDTA (10 mM) had no effect on its haemagglutinating activity. PAS reagent staining showed that the lectin was not a glycoprotein. The sub-acute studies on rats showed elevated levels of ALT, AST, serum bilirubin, total protein in serum and liver homogenates suggesting damage to liver and spleen. The study concluded that the aerial bulb of D. bulbifera lectin was non-specific in its heamagglutinating activity and dimeric in its structure. The lectin shared some physicochemical characteristics with lectins from other Dioscorecea species and was moderately toxic to the liver and spleen of treated animals.

Keywords: Dioscorea bulbifera, heamagglutinin, lectin, toxicity

Procedia PDF Downloads 127

Authors: Rowan Moore, Kai Scheffler, Eugen Damoc, Jennifer Sutton, Aaron Bailey, Stephane Houel, Simon Cubbon, Jonathan Josephs

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Purpose: MS analysis of monoclonal antibodies (mAbs) at the protein and peptide levels is critical during development and production of biopharmaceuticals. The compositions of current generation therapeutic proteins are often complex due to various modifications which may affect efficacy. Intact proteins analyzed by MS are detected in higher charge states that also provide more complexity in mass spectra. Protein analysis in native or native-like conditions with zero or minimal organic solvent and neutral or weakly acidic pH decreases charge state value resulting in mAb detection at higher m/z ranges with more spatial resolution. Methods: Three commercially available mAbs were used for all experiments. Intact proteins were desalted online using size exclusion chromatography (SEC) or reversed phase chromatography coupled on-line with a mass spectrometer. For streamlined use of the LC- MS platform we used a single SEC column and alternately selected specific mobile phases to perform separations in either denaturing or native-like conditions: buffer A (20 % ACN, 0.1 % FA) with Buffer B (100 mM ammonium acetate). For peptide analysis mAbs were proteolytically digested with and without prior reduction and alkylation. The mass spectrometer used for all experiments was a commercially available Thermo Scientific™ hybrid Quadrupole-Orbitrap™ mass spectrometer, equipped with the new BioPharma option which includes a new High Mass Range (HMR) mode that allows for improved high mass transmission and mass detection up to 8000 m/z. Results: We have analyzed the profiles of three mAbs under reducing and native conditions by direct infusion with offline desalting and with on-line desalting via size exclusion and reversed phase type columns. The presence of high salt under denaturing conditions was found to influence the observed charge state envelope and impact mass accuracy after spectral deconvolution. The significantly lower charge states observed under native conditions improves the spatial resolution of protein signals and has significant benefits for the analysis of antibody mixtures, e.g. lysine variants, degradants or sequence variants. This type of analysis requires the detection of masses beyond the standard mass range ranging up to 6000 m/z requiring the extended capabilities available in the new HMR mode. We have compared each antibody sample that was analyzed individually with mixtures in various relative concentrations. For this type of analysis, we observed that apparent native structures persist and ESI is benefited by the addition of low amounts of acetonitrile and formic acid in combination with the ammonium acetate-buffered mobile phase. For analyses on the peptide level we analyzed reduced/alkylated, and non-reduced proteolytic digests of the individual antibodies separated via reversed phase chromatography aiming to retrieve as much information as possible regarding sequence coverage, disulfide bridges, post-translational modifications such as various glycans, sequence variants, and their relative quantification. All data acquired were submitted to a single software package for analysis aiming to obtain a complete picture of the molecules analyzed. Here we demonstrate the capabilities of the mass spectrometer to fully characterize homogeneous and heterogeneous therapeutic proteins on one single platform. Conclusion: Full characterization of heterogeneous intact protein mixtures by improved mass separation on a quadrupole-Orbitrap™ mass spectrometer with extended capabilities has been demonstrated.

Keywords: disulfide bond analysis, intact analysis, native analysis, mass spectrometry, monoclonal antibodies, peptide mapping, post-translational modifications, sequence variants, size exclusion chromatography, therapeutic protein analysis, UHPLC

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Authors: Augustine O. Ani, Ifeyinwa E. Ezemagu, Eunice A. Akuru

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A seven-week study was conducted to evaluate the response of grower turkeys to varying dietary levels of Moringa oleifera leaf meal (MOLM) in a humid tropical environment. A total of 90 twelve weeks old male and female grower turkeys were randomly divided into five groups of 18 birds each in a completely randomized design (CRD) and assigned to five caloric (2.57-2.60 Mcal/kg ME) and isonitrogenous (19.95% crude protein) diets containing five levels (0, 15, 20, 25 and 30%) of MOLM, respectively. Each treatment was replicated three times with 6 birds per replicate housed in a deep litter pen of fresh wood shavings measuring 1.50m x 1.50m. Feed and water were provided to the birds' ad libitum. Parameters measured were: final live weight (FLW) daily weight gain (DWG), daily feed intake (DFI), feed conversion ratio (FCR), protein efficiency ratio (PER), packed cell volume (PCV), haemoglobin concentration (Hb), red blood cell (RBC) count, white blood cell (WBC) count, mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC), feed cost / kg weight gain and apparent nutrient retention. Results showed that grower turkeys fed 20% MOLM diet had significantly (p < 0.05) higher FLW and DWG values (4410.30 g and 34.49 g, respectively) and higher DM and NFE retention values (67.28 and 58.12%, respectively) than turkeys fed other MOLM diets. Feed cost per kg gain decreased significantly (p < 0.05) with increasing levels of MOLM in the diets. The PCV, Hb, WBC, MCV, MCH and MCHC values of grower turkeys fed 20% MOLM diet were significantly (p < 0.05) higher than those of grower turkeys fed other diets. It was concluded that a diet containing 20% MOLM is adequate for the normal growth of grower turkeys in the tropics.

Keywords: Diets, grower turkeys, Moringa oleifera leaf meal, response, tropical environment

Procedia PDF Downloads 144

Authors: Johanrik, Arini Apriliani, Fikri Haikal, Dias Yuca, Muhammad Abdul Latif, Edijanti Goenarwo, Nurita Pratama Sari

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Diarrhea is one of the leading causes of morbidity and mortality in many countries, as well as responsible for the deaths of millions of people each year. Previous research showed that the leaves, bark, and root bark of kedondong contains saponins, tannins, and flavonoids. Tannins have anti-diarrheal effects that work as the freeze of protein/astringent, and may inhibit the secretion of chloride over the tannate bonding between protein in the intestines. Chemical compounds of flavonoids also have an effect as anti-diarrheal block receptors Cl ˉ in intestinal thus reducing the secretion of Cl ˉ to the intestinal lume .This research aims to know the anti-diarrheal activity of extracts kedondong leaf in mice Balb/C strain males in vivo. This research also proves kedondong leaves as an anti-diarrhea through trial efficacy of kedondong leaves as antisekretori and antimotilitas. This research using post-test only controlled group design. Analysis of statistical data normality and homogenity were tested by Kolmogorov Smirnov. If the data obtained homogenous then using ANOVA test. This research using ethanolic extracts kedondong leaf 200, 400 and 800 mg/kgBW to prove there is anti-diarrhea it makes into six treatment groups, for anti-secretory it makes into five treatment groups and anti-motility became five treatment groups. The result showed dose of ethanolic extracts kedondong leaf 800 mg/kgBW have significant value (p<0.005). The conclusion from this extracts kedondong leaf research 800 mg/kgBW have pharmacological effects as antidiarrhea on Balb/C strain male mice with a mechanism of action as anti-secretory and anti-motility.

Keywords: anti-diarrhea, anti-secretory, anti-motility, kedondong leaf

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Authors: Ximena Calle, Plinio Cantero-López, Felipe Muñoz-Córdova, Mayarling-Francisca Troncoso, Sergio Lavandero, Valentina Parra

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MUL1, a mitochondrial E3 ubiquitin ligase anchored to the outer mitochondrial membrane, is highly expressed in the heart. MUL1 is involved in multiple biological pathways associated with mitochondrial dynamics. Increased MUL1 affects the balance between fission and fusion, affecting mitochondrial function, which plays a crucial role in myocardial function. Therefore, it is interesting to evaluate the effect of cardiac-specific overexpression of MUL1 on myocardial function. Aim: To determine heart functionality in a mouse model with cardio-specific overexpression MUL1 protein. Methods and Results: Male C57BL/Tg transgenic mice with cardiomyocyte-specific overexpression of MUL1 (n=10) and control (n=4) were evaluated at 12, 27, and 35 weeks of age. Glucose tolerance curve determination was performed after a 6-hours fast to assess metabolic capacity, treadmill test, and systolic, and diastolic pressure was evaluated by the mouse tail-cuff blood pressure system equipment. The result showed no glucose tolerance curve, and the treadmill test demonstrated no significant changes between groups. However, substantial changes in diastolic function were observed by ultrasound and determination of cardiac hypertrophy proteins by western blot. Conclusions: Cardio-specific overexpression of MUL1 in mice without any treatment affects diastolic cardiac function, thus showing the important role contributed by MUL1 in the heart. Future research should evaluate the effect of cardiomyocyte-specific overexpression of MUL1 in pathological conditions such as a high-fat diet is one of the main risk factors for cardiovascular disease.

Keywords: diastolic dysfunction, hypertrophy cardiac, mitochondrial E3 ubiquitin ligase 1, MUL1

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Authors: Devinder Kaur Sugga, Ekamdeep Kaur, Jaspreet Kaur, C. Rajesh, Preeti Rajesh, Harsimran Kaur

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Withanolides are steroidal lactones and are highly oxygenated phytoconstituents that can be developed as potential anti-carcinogenic agents. The two main withanolides, namely Withaferin A and Withanolides D, have been extensively studied for their pharmacological activities. Both these withanolides are present in the Withania somnifera (WS) leaves belonging to the family Solanaceae, also known as “Indian ginseng .”In this study effects of WS leaf extract on the MCF7 breast cancer cell line were investigated by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effects and in vitro wound-healing assay to study the effect on cancer cell migration. Our data suggest WS extracts have cytotoxic effects and are effective anti-migrating agents and thus can be a source of potential candidates for the development of potential agents against metastasis. Thus, it can be a source of potential candidates for the development of potential agents against metastasis. Insight into these results, the in-silico approach to identify the possible protein targets interacting with withanolides was taken. Protein kinase C alpha (PKCα) was among the selected 5 top-ranked target proteins identified by the Swiss Target Prediction tool. PKCα is known to promote the growth and invasion of cancer cells and is being evaluated as a prognostic biomarker and therapeutic target in clinically aggressive tumors. Molecular docking of Withaferin A and Withanolides D was performed using AutoDock Vina. Both the bioactive compounds interacted with PKCα. The targets predicted using this approach will serve as leads for the possible therapeutic potential of withanolides, the bioactive ingredients of WS extracts, as anti-cancer drugs.

Keywords: withania somnifera, withaferin A, withanolides D, PKCα

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Authors: Susmita Roy, Madhavan Nallani

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In recent years' polymersomes, self-assembled polymeric vesicles emerge from block copolymers, have been widely investigated due to their enhance stability and unique advantageous properties compared to their phospholipid counterpart, liposomes, dendrimers, and micelles. It provides a distinctive platform for advanced therapeutics and the creation of complex (bio) catalytically active systems for research in Nanomedicine and synthetic biology. Inspired by nature, where compartmentalization of biological components is all ubiquitous, we are interested in developing a platform technology of self-assembled multifunctional compartments with applications in areas from targeted drug/gene delivery, biosensing, pharmaceutical to cosmetics. Polymersome surfaces can be a proper choice of derivatization with a controlled amount of functional groups. To achieve site-specific targeting of polymersomes, biological recognition motives can be attached to the polymersomes surface by standard bioconjugation techniques, (like esterification, amidation, thiol-maleimide coupling, click-chemistry routes or other coupling methods). Herein, we are developing easy going, one-step bioconjugation strategies for site-specific surface functionalized biodegradable polymeric and/or polymer-lipid hybrid vesicles for targeted drug delivery. Biodegradable polymer, polycaprolactone-b-polyethylene glycol (PCL-PEG), polylactic acid-b-polyethylene glycol (PLA-PEG) and phospholipid, 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC) has been widely used for numerous vesicle formulations. Some of these drug-loaded formulations are being tested on mice for controlled release. These surface functionalized polymersomes are also appropriate for membrane protein reconstitution/insertion, antibodies conjugation and various bioconjugation with diverse targeted molecules for controlled drug delivery.

Keywords: drug delivery, membrane protein, polymersome, surface modification

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Authors: Bipasa Dey, Varsha Panwar, Tanmay Dutta

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Laccase, a multicopper oxidase (EC 1.10.3.2) have been at the forefront as a superior industrial biocatalyst. They are versatile in terms of bestowing sustainable and ecological catalytic reactions such as polymerisation, xenobiotic degradation and bioremediation of phenolic and non-phenolic compounds. Regardless of the wide biotechnological applications, the critical limiting factors viz. reusability, retrieval, and storage stability still prevail. This can cause an impediment in their applicability. Crosslinked enzyme aggregates (CLEAs) have emerged as a promising technique that rehabilitates these essential facets, albeit at the expense of their enzymatic activity. The carrier free crosslinking method prevails over the carrier-bound immobilisation in conferring high productivity, low production cost owing to the absence of additional carrier and circumvent any non-catalytic ballast which could dilute the volumetric activity. To the best of our knowledge, the ε-amino group of lysyl residue is speculated as the best choice for forming Schiff’s base with glutaraldehyde. Despite being most preferrable, excess glutaraldehyde can bring about disproportionate and undesirable crosslinking within the catalytic site and hence could deliver undesirable catalytic losses. Moreover, the surface distribution of lysine residues in Trametes versicolor laccase is significantly less. Thus, to mitigate the adverse effect of glutaraldehyde in conjunction with scaling down the degradation or catalytic loss of the enzyme, crosslinking with inert substances like gelatine, collagen, Bovine serum albumin (BSA) or excess lysine is practiced. Analogous to these molecules, sugars have been well known as a protein stabiliser. It helps to retain the structural integrity, specifically secondary structure of the protein during aggregation by changing the solvent properties. They are comprehended to avert protein denaturation or enzyme deactivation during precipitation. We prepared crosslinked enzyme aggregates (CLEAs) of laccase from T. versicolor with the aid of sugars. The sugar CLEAs were compared with the classic BSA and glutaraldehyde laccase CLEAs concerning physico-chemical properties. The activity recovery for the fructose CLEAs were found to be ~20% higher than the non-sugar CLEA. Moreover, the 𝐾𝑐𝑎𝑡𝐾𝑚⁄ values of the CLEAs were two and three-fold higher than BSA-CLEA and GACLEA, respectively. The half-life (t1/2) deciphered by sugar-CLEA was higher than the t1/2 of GA-CLEAs and free enzyme, portraying more thermal stability. Besides, it demonstrated extraordinarily high pH stability, which was analogous to BSA-CLEA. The promising attributes of increased storage stability and recyclability (>80%) gives more edge to the sugar-CLEAs over conventional CLEAs of their corresponding free enzyme. Thus, sugar-CLEA prevails in furnishing the rudimentary properties required for a biocatalyst and holds many prospects.

Keywords: cross-linked enzyme aggregates, laccase immobilization, enzyme reusability, enzyme stability

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Authors: Svitlana Antonenko, Gennady Telegeev

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Introductive statement: The development of chronic myeloid leukemia (CML) is caused by Bcr-Abl oncoprotein. Modern treatments with tyrosine kinase inhibitors are greatly complicated by the mutational variability of the Bcr-Abl oncoprotein, which causes drug resistance. Therefore, there is an urgent need to develop new approaches to the treatment of the disease, which will allow modeling the level of Bcr-Abl oncoprotein in the cell. Promising in this direction is the identification of proteases that can selectively promote cellular proteolysis of oncoproteins. The aim of the study was to study the effect of the interaction of Bcr-Abl with deubiquitinase USP1 on the level of oncoprotein in CML cells. Methodology: K562 cells were selected for the experiment. Сells were incubated with ML323 inhibitor for 24 hours. Precipitation of endogenous proteins from K562 cell lysate was performed using anti-Bcr-Abl antibodies. Cell lysates and precipitation results were studied by Western blot. Subcellular localization of proteins was studied by immunofluorescence analysis followed by confocal microscopy. The results were analyzed quantitatively and statistically. Major findings: The Bcr-Abl/USP1 protein complex was detected in CML cells, and it was found that inhibition of USP1 deubiquitinating activity by the compound ML323 leads to disruption of this protein complex and a decrease in the level of Bcr-Abl oncoprotein in cells. The interaction of Bcr-Abl with USP1 may result in deubiquitination of the oncoprotein, which disrupts its proteasomal degradation and leads to the accumulation of CML in cells. Conclusion: We believe that the interaction of oncoprotein with USP1 may be one of the prerequisites that contribute to malignant cell transformation due to the deubiquitination of oncoprotein, which leads to its accumulation and disease progression. A correlation was found between the deubiquitinating activity of USP1 and the level of oncoprotein in CML cells. Thus, we identify deubiquitinase USP1 as a promising therapeutic target for the development of a new strategy for the treatment of CML by modulating the level of Bcr-Abl in the cell.

Keywords: chronic myeloid leukemia, Bcr-Abl, USP1, deubiquitination Bcr-Abl, K562 cell

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Authors: Sina Mahdavi

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Background and Objective: Multiple sclerosis (MS) is an inflammatory autoimmune disease of the CNS that affects the myelination process in the central nervous system (CNS). Complex interactions of various "environmental or infectious" factors may act as triggers in autoimmunity and disease progression. The association between viral infections, especially human endogenous retrovirus (HERV) and MS is one potential cause that is not well understood. This study aims to summarize the available data on HERV infection in MS disease progression. Materials and Methods: For this study, the keywords "Multiple sclerosis", "Human endogenous retrovirus", and "central nervous system" in the databases PubMed, Google Scholar, Sid, and MagIran between 2016 and 2022 were searched and 14 articles chosen, studied, and analyzed. Results: In the leptomeningeal cells of MS patients, a retrovirus-like element associated with reverse transcriptase (RT) activity called multiple sclerosis-associated retroviruses (MSRV) has been identified. HERVs are expressed in the human CNS despite mechanisms to suppress their expression. External factors, especially viral infections such as influenza virus, Epstein-Barr virus, and herpes simplex virus type 1, can activate HERV gene expression. The MSRV coat protein is activated by activating TLR4 at the brain surface, particularly in oligodendroglial progenitor cells and macrophages, leading to immune cascades followed by the downregulation of myelin protein expression. The HERV-K18 envelope gene (env) acts as a superantigen and induces inflammatory responses in patients with MS. Conclusion: There is a high expression of endogenous retroviruses during the course of MS, which indicates the relationship between HERV and MS, that this virus can play a role in the development of MS by creating an inflammatory state. Therefore, measures to modulate the expression of endogenous retroviruses may be effective in reducing inflammatory processes in demyelinated areas of MS patients.

Keywords: multiple sclerosis, human endogenous retrovirus, central nervous system, MSRV

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Authors: Bernice Lottering, Yi-Wen Lin

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Chronic pain and depression have an estimated 80% rate of comorbidity with unsatisfactory treatment interventions signifying the importance of developing effective therapeutic interventions for a serious chronic condition affecting a large majority of the global population. Chronic pain is defined as persistent pain presenting for over 3 months. This disease state increases the risk of developing depression in comparison to healthy individuals. In the current study, complete Freund’s adjuvant (CFA) was used to induce cell-mediated chronic inflammatory pain in a murine model. Significant mechanical and thermal hyperalgesia was induced, alongside observable depression-like behaviors. These conditions were attenuated through the use of electroacupuncture (EA). Similarly, these effects were also investigated with respect to the transient receptor potential vanilloid 1 (TRPV1), by analyzing the effects of TRPV1 gene deletion on the comorbidity of chronic pain and depression. The expression of the TRPV1 inflammatory response, and related downstream molecules, including protein kinases (PKs), mitogen-activated protein kinase (MAPKs), and transcriptional factors, were significantly reduced in the thalamus, prefrontal cortex (PFC), hippocampus, and periaqueductal gray (PAG) of CFA-treated mice. In addition, phosphorylated N-methyl-D-aspartate (NMDA) receptor 1 was also found to be reduced in the aforementioned areas, suggesting potential application and validity in a clinical setting. Our study determined the prospective therapeutic effects of EA in the treatment of chronic inflammatory pain and depression comorbidity and provides a novel and detailed mechanism underlying EA-mediated analgesia. These findings may be relevant in the utilization of clinical intervention approaches related to chronic pain and depression comorbidity.

Keywords: chronic pain, depression, NMDA, prefrontal cortex, TRPV1

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Authors: Salma A. El-Marasy, Reham M. Abd-Elsalam, Omar A. Ahmed-Farid

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Dementia is characterized by impairments in memory and other cognitive abilities. This study aims to elucidate the possible ameliorative effect of silymarin on scopolamine-induced dementia using the object recognition test (ORT). The study was extended to demonstrate the role of cholinergic activity, oxidative stress, neuroinflammation, brain neurotransmitters and histopathological changes in the anti-amnestic effect of silymarin in demented rats. Wistar rats were pretreated with silymarin (200, 400, 800 mg/kg) or donepezil (10 mg/kg) orally for 14 consecutive days. Dementia was induced after the last drug administration by a single intraperitoneal dose of scopolamine (16 mg/kg). Then behavioral, biochemical, histopathological, and immunohistochemical analyses were then performed. Rats pretreated with silymarin counteracted scopolamine-induced non-spatial working memory impairment in the ORT and decreased acetylcholinesterase (AChE) activity, reduced malondialdehyde (MDA), elevated reduced glutathione (GSH), restored gamma-aminobutyric acid (GABA) and dopamine (DA) contents in the cortical and hippocampal brain homogenates. Silymarin dose-dependently reversed scopolamine-induced histopathological changes. Immunohistochemical analysis showed that silymarin dose-dependently mitigated protein expression of a glial fibrillary acidic protein (GFAP) and nuclear factor kappa-B (NF-κB) in the brain cortex and hippocampus. All these effects of silymarin were similar to that of the standard anti-amnestic drug, donepezil. This study reveals that the ameliorative effect of silymarin on scopolamine-induced dementia in rats using the ORT maybe in part mediated by, enhancement of cholinergic activity, anti-oxidant and anti-inflammatory activities as well as mitigation in brain neurotransmitters and histopathological changes.

Keywords: dementia, donepezil, object recognition test, rats, silymarin, scopolamine

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Authors: Yu-Chieh Chen, Li-Yun Wang, Chi-Chih Chen, Huy Hùng Đào, Ya-Mei Chen, Ming-Chu Cheng, Wen-Bin Chung, Hso-Chi Chaung, Guan-Ming Ke

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Many recent researches have demonstrated that CD154, a protein primarily expressed on activated T cell molecules, has potentially acted as a molecular adjuvant to improve the immunogenicity of subunit vaccines against viral infections. Classical swine fever (CSF) affects the swine industry worldwide that is one of the most devastating and highly contagious pig diseases. It is listed by the World Organization for Animal Health (OIE) as an infectious animal disease that must be reported. Although pigs vaccinated with subunit vaccines can be differentially diagnosed from those infected animals, subunit vaccines usually need adjuvants to enhance and elicit immune responses. In this study, CD154 was linked with CSFV E2 sequences and then expressed in CHO cells to produce the fusion protein as E2-CD154. The porcine specific CpG adjuvant was also used in one of the formulations. The specific pathogen-free pigs (SPF) at the age of 4-week-old were randomly separated into four groups, vaccinated with E2-CpG, E2-CD154, E2-CD154-CpG or the commercial Bayovac® CSF-E2 vaccine and boosted two weeks after primary vaccination. The results showed that the percentages of CD4+ and CD4+IL2+ in peripheral blood mononuclear cells (PBMC) in E2-CD154 vaccinated piglets seven days after primary vaccination were gained by 1-5% relative to the control group. In addition, the percentages of CD4+IFNγ+ T cells had slightly edged up 0.1-0.3% compared with the control group. Also, increased E2-specific IFNγ levels had edged up CD4+CD8+ T cells found in E2-CD154 and E2-CD154-CpG groups, particularly in the E2-CD154-CpG group. These results implicate that CD154 may enhance cellular immunity and synergistically act with species-specific CpG adjuvant as a dual-phase adjuvant. Therefore, the CD154 may be beneficial as a promising adjuvant in subunit vaccines.

Keywords: CD154, CpG adjuvant, cellular immunity, subunit vaccine, pig

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Authors: Manal E. A Elhalwagy, Nadia Amin Abdulmajeed, Hanan S. Alnahdi, Enas N. Danial

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Baron is herbicide includes (48% glyphosate) widely used in Egypt. The present study assesses the cytotoxic and genotoxic effect of baron on rats liver. Two groups of rats were treated orally with 1/10 LD 50, (275.49 mg kg -1) and 1/40 LD 50, (68.86 mg kg-1) glyphosate for 28 days compared with control group. Serum and liver tissues were taken at 14 and 28 days of treatment. An inhibition in Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were recorded at both treatment periods and reduction in total serum protein (TP) and albumin (ALB). However, non-significant changes in serum acetylcholinesterase (AChE). Elevation in oxidative stress biomarker malondyaldehyde (MDA) and the decline in detoxification biomarker total reduced glutathione (GSH), Glutathione S-transferase (GST) and superoxide dismutase (SOD) in liver tissues led to increase in percentage of DNA damage. Destruction in liver tissue architecture was observed . Although, Baron was classified in the safe category pesticides repeated exposure to small doses has great danger effect.

Keywords: glyphosate, liver toxicity, oxidative stress, DNA damage, commet assay

Procedia PDF Downloads 383

Authors: K. Shailaja

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The advanced glycation end products (AGEs) formed between the reducing sugar and protein as a result of Oxidative stress and non-enzymatic glycosylation play an important role in pathogenesis of diabetes and aging complication. Glycation results in the production of free radicals. The oxidation process is believed to play an important role in AGEs formation. Thus agents with antioxidative property and antiglycation activity may retard the process of AGEs formation. Selected medicinal plants for the present study include Catharanthus roseus, Bougainvillea spectabilis (pink flowers), Cinnamomum tamala, Cinnamomum zeylanica, Abutilon indicum, Asparagus racemosus, and Sapindus emarginatus. The crude ethanolic extracts of the selected medicinal plants at varying concentrations ranging from 1-100 mg/ml were evaluated for in vitro antioxidant and protein glycation activities by FRAP and glucose-BSA assay respectively. Among all the plants tested, Bougainvillea spectabilis, Catharanthus roseus and Abutilon indicum showed strong antioxidant activity The antioxidant activity was expressed as mg of Gallic acid/ gm sample which was found to be 4.3 mg, 1.3mg, and 1.3mg respectively for Bougainvillea spectabilis, Catharanthus roseus and Abutilon indicum. The results of inhibition of the initial glycation product i.e., fructosamine was found to be 35% for Asparagus racemosus, Cinnamomum tamala and Abutilon indicum followed by the other plant extracts. The results indicate that these plants are potential sources of natural antioxidants which have free radical scavenging activity and might be used not only for reducing oxidative stress in diabetes but also open a new research avenues in the field of Natural Products.

Keywords: in vitro antioxidant activity, anti-glycation activity, ethanol extracts, polyphenols, Catharanthus roseus, Cinnamomum tamala

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Authors: Z. Karahaliloğlu, E. Yalçın, M. Demirbilek, E.B. Denkbaş

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Critical-sized bone defects caused by trauma, bone diseases, prosthetic implant revision or tumor excision cannot be repaired by physiological regenerative processes. Current orthopedic applications for critical-sized bone defects are to use autologous bone grafts, bone allografts, or synthetic graft materials. However, these strategies are unable to solve completely the problem, and motivate the development of novel effective biological scaffolds for tissue engineering applications and regenerative medicine applications. In particular, scaffolds combined with a variety of bio-agents as fundamental tools emerge to provide the regeneration of damaged bone tissues due to their ability to promote cell growth and function. In this study, a magnetic silk fibroin (SF) hydrogel scaffold was prepared by electrogelation process of the concentrated Bombxy mori silk fibroin (8 %wt) aqueous solution. For enhancement of osteoblast-like cells (SaOS-2) growth and adhesion, basal fibroblast growth factor (bFGF) were conjugated physically to the HSA-coated magnetic nanoparticles (Fe3O4) and magnetic SF e-gel scaffolds were prepared by incorporation of Fe3O4, HSA (human serum albumin)=Fe3O4 and HSA=Fe3O4-bFGF nanoparticles. HSA=Fe3O4, HSA=Fe3O4-bFGF loaded and bare SF e-gels scaffolds were characterized using scanning electron microscopy (SEM.) For cell studies, human osteoblast-like cell line (SaOS-2) was used and an MTT assay was used to assess the cytotoxicity of magnetic silk fibroin e-gel scaffolds and cell density on these surfaces. For the evaluation osteogenic activation, ALP (alkaline phosphatase), the amount of mineralized calcium, total protein and collagen were studied. Fe3O4 nanoparticles were successfully synthesized and bFGF was conjugated to HSA=Fe3O4 nanoparticles with %97.5 of binding yield which has a particle size of 71.52±2.3 nm. Electron microscopy images of the prepared HSA and bFGF incorporated SF e-gel scaffolds showed a 3D porous morphology. In terms of water uptake results, bFGF conjugated HSA=Fe3O4 nanoparticles has the best water absorbability behavior among all groups. In the in-vitro cell culture studies realized using SaOS-2 cell line, the coating of Fe3O4 nanoparticles surface with a protein enhance the cell viability and HSA coating and bFGF conjugation, the both have an inductive effect in the cell proliferation. One of the markers of bone formation and osteoblast differentiation, according to the ALP activity and total protein results, HSA=Fe3O4-bFGF loaded SF e-gels had significantly enhanced ALP activity. Osteoblast cultured HSA=Fe3O4-bFGF loaded SF e-gels deposited more calcium compared with SF e-gel. The proposed magnetic scaffolds seem to be promising for bone tissue regeneration and used in future work for various applications.

Keywords: basic fibroblast growth factor (bFGF), e-gel, iron oxide nanoparticles, silk fibroin

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Authors: Maher J. Tadros

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This research was conducted to study the effect of different salinity concentrations on the plant water relation and forage quality on two multipurpose forest trees species seedlings Leucaena leucocephala (Lam.) de wit and Acacia saligna (Labill.). Five different salinity concentrations mixture between sodium chloride and calcium chloride (v/v, 1:1) were applied. The control (Distilled Water), 2000, 4000, 6000, and 8000 ppm were used to water the seedlings for 3 months. The research results presented showed a marked variation among the two species in response to salinity. The Leucaena was able to withstand the highest level of salinity compared to Acacia all over the studied parameters except in the relative water content. Although all the morphological characteristics studied for the two species showed a marked decrease under the different salinity concentrations, except the shoot/root ratio that showed a trend of increase. The water stress measure the leaf water potential was more negative with as the relative water content increase under that saline conditions compared to the control. The forage quality represented by the crude protein and nitrogen content were low at 6000 ppm compared to the 8000 ppm in L. Leucocephala that increased compared that level in A. saligna. Also the results showed that growing both Leucaena and Acacia provide a good source of forage when that grow under saline condition which will be of great benefits to the agricultural sector especially in the arid and semiarid areas were these species can provide forage with high quality forage all year around when grown under irrigation with saline. This research recommended such species to be utilized and grown for forages under saline conditions.

Keywords: plant water relations, growth performance, salinity stress, protein content, forage quality, multipurpose trees

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Authors: M. Naimi, M. B. Khaled

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The present study consisted of an applied test in meat system to assess the effectiveness of three bio agents bacteriocinproducing strains: Lm24: Lactobacillus sakei, Lm14and Lm25: Pediococcus spp. Two tests were carried out: The ex-situ test was intended for three batches added with crude bacteriocin solutions at 12.48 AU/ml for Lm25 and 8.4 AU/ml for Lm14 and Lm24. However, the in situ one consisted of four batches; three of them inoculated with one bacteriocinogenic Lm25, Lm14, Lm24, respectively. The fourth one was used in mixture: Lm14+m24 at approximately of 107 CFU/ml. The two used tests were done in the presence of the pathogen St. aureus ATCC 6538, as a test strain at 103 CFU/ml. Another batch served as a positive or a negative control was used too. The incubation was performed at 7°C. Total viable counts, staphylococci and lactic acid bacteria, at the beginning and at selected times with interval of three days were enumerated. Physicochemical determinations (except for in situ test): pH, dry mater, sugars, fat and total protein, at the beginning and at end of the experiment, were done, according to the international norms. Our results confirmed the ex situ effectiveness. Furthermore, the batches affected negatively the total microbial load over the incubation days, and showed a significant regression in staphylococcal load at day seven, for Lm14, Lm24, and Lm25 of 0.73, 2.11, and 2.4 log units. It should be noticed that, at the last day of culture, staphylococcal load was nil for the three batches. In the in situ test, the cultures displayed less inhibitory attitude and recorded a decrease in staphylococcal load, for Lm14, Lm24, Lm25, Lm14+m24 of 0.73, 0.20, 0.86, 0.032 log units. Therefore, physicochemical analysis for Lm14, Lm24, Lm25, Lm14+m24 showed an increase in pH from 5.50 to 5.77, 6.18, 5.96, 7.22, a decrease in dry mater from 7.30% to 7.05%, 6.87%, 6.32%, 6.00%.This result reflects the decrease in fat ranging from 1.53% to 1.49%, 1.07%, 0.99%, 0.87%; and total protein from 6.18% to 5.25%, 5.56%, 5.37%, 5.5%. This study suggests that the use of selected strains as Lm25 could lead to the best results and would help in preserving and extending the shelf life of lamb meat.

Keywords: biocontrol, in situ, ex situ, meat system, St. aureus, Lactobacillus sakei, Pediococcus spp.

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Authors: N. Umasuthan, S. D. N. K. Bathige, W. S. Thulasitha, I. Whang, J. Lee

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The MyD88 is an evolutionarily conserved host-expressed adaptor protein that is essential for proper TLR/ IL1R immune-response signaling. A previously identified complete cDNA (1626 bp) of OfMyD88 comprised an ORF of 867 bp encoding a protein of 288 amino acids (32.9 kDa). The gDNA (3761 bp) of OfMyD88 revealed a quinquepartite genome organization composed of 5 exons (with the sizes of 310, 132, 178, 92 and 155 bp) separated by 4 introns. All the introns displayed splice signals consistent with the consensus GT/AG rule. A bipartite domain structure with two domains namely death domain (24-103) coded by 1st exon, and TIR domain (151-288) coded by last 3 exons were identified through in silico analysis. Moreover, homology modeling of these two domains revealed a similar quaternary folding nature between human and rock bream homologs. A comprehensive comparison of vertebrate MyD88 genes showed that they possess a 5-exonic structure. In this structure, the last three exons were strongly conserved, and this suggests that a rigid structure has been maintained during vertebrate evolution. A cluster of TATA box-like sequences were found 0.25 kb upstream of cDNA starting position. In addition, putative 5'-flanking region of OfMyD88 was predicted to have TFBS implicated with TLR signaling, including copies of NFB1, APRF/ STAT3, Sp1, IRF1 and 2 and Stat1/2. Using qPCR technique, a ubiquitous mRNA expression was detected in liver and blood. Furthermore, a significantly up-regulated transcriptional expression of OfMyD88 was detected in head kidney (12-24 h; >2-fold), spleen (6 h; 1.5-fold), liver (3 h; 1.9-fold) and intestine (24 h; ~2-fold) post-Fla challenge. These data suggest a crucial role for MyD88 in antibacterial immunity of teleosts.

Keywords: MyD88, innate immunity, flagellin, genomic analysis

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Authors: Deniz Dincel, Sena Ardicli, Hale Samli, Mustafa Ogan, Faruk Balci

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The aim of the study was to determine the effects of some environmental and genetic factors on growth, fertility traits, milk yield and composition in Saanen goats. For this purpose, the total of 173 Saanen goats and kids were investigated for growth, fertility and milk traits in Marmara Region of Turkey. Fertility parameters (n=70) were evaluated during two years. Milk samples were collected during the lactation and the milk yield/components (n=59) of each goat were calculated. In terms of CSN3 and AGPAT6 gene; the genotypes were defined by PCR-RFLP. Saanen kids (n=86-112) were measured from birth to 6 months of life. The birth, weaning, 60ᵗʰ, 90ᵗʰ, 120ᵗʰ and 180tᵗʰ days of average live weights were calculated. The effects of maternal age on pregnancy rate (p < 0.05), birth rate (p < 0.05), infertility rate (p < 0.05), single born kidding (p < 0.001), twinning rate (p < 0.05), triplet rate (p < 0.05), survival rate of kids until weaning (p < 0.05), number of kids per parturition (p < 0.01) and number of kids per mating (p < 0.01) were found significant. The impacts of year on birth rate (p < 0.05), abortion rate (p < 0.001), single born kidding (p < 0.01), survival rate of kids until weaning (p < 0.01), number of kids per mating (p < 0.01) were found significant for fertility traits. The impacts of lactation length on all milk yield parameters (lactation milk, protein, fat, totally solid, solid not fat, casein and lactose yield) (p < 0.001) were found significant. The effects of age on all milk yield parameters (lactation milk, protein, fat, total solid, solid not fat, casein and lactose yield) (p < 0.001), protein rate (p < 0.05), fat rate (p < 0.05), total solid rate (p < 0.01), solid not fat rate (p < 0.05), casein rate (p < 0.05) and lactation length (p < 0.01), were found significant too. However, the effect of AGPAT6 gene on milk yield and composition was not found significant in Saanen goats. The herd was found monomorphic (FF) for CSN3 gene. The effects of sex on live weights until 90ᵗʰ days of life (birth, weaning and 60ᵗʰ day of average weight) were found significant statistically (p < 0.001). The maternal age affected only birth weight (p < 0,001). The effects month at birth on all of the investigated day [the birth, 120ᵗʰ, 180ᵗʰ days (p < 0.05); the weaning, 60ᵗʰ, 90ᵗʰ days (p < 0,001)] were found significant. The birth type was found significant on the birth (p < 0,001), weaning (p < 0,01), 60ᵗʰ (p < 0,01) and 90ᵗʰ (p < 0,01) days of average live weights. As a result, screening the other regions of CSN3, AGPAT6 gene and also investigation the phenotypic association of them should be useful to clarify the efficiency of target genes. Environmental factors such as maternal age, year, sex and birth type were found significant on some growth, fertility and milk traits in Saanen goats. So consideration of these factors could be used as selection criteria in dairy goat breeding.

Keywords: fertility, growth, milk yield, Saanen goats

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