Search results for: mitochondrial proteins

Authors: Hye Kyung Kim, Myung-Gyou Kim, Kang-Hyun Leem

Abstract:

Postmenopausal osteoporosis is characterized by low bone density which leads to increased bone fragility and greater susceptibility to fracture. Current treatments for osteoporosis are dominated by drugs that inhibit bone resorption although they also suppress bone formation that may contribute to pathogenesis of osteonecrosis. To restore the extensive bone loss, there is a great need for anabolic treatments that induce osteoblasts to build new bone. Pre-osteoblastic cells produce proteins of the extra-cellular matrix, including type I collagen at first, and then to successively produce alkaline phosphatase (ALP) and osteocalcin during differentiation to osteoblasts. Finally, osteoblasts deposit calcium. Present study investigated the effects of egg yolk peptide (EYP) on osteogenic activities and bone matrix gene expressions in human osteoblastic MG-63 cells. The effects of EYP on cell proliferation, alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization were measured. The expression of osteogenic genes including COL1A1 (collagen, type I, alpha 1), ALP, BGLAP (osteocalcin), and SPP1 (secreted phosphoprotein 1, osteopontin) were measured by quantitative realtime PCR. EYP dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. Furthermore, COL1A1, ALP, and SPP1 gene expressions were increased by EYP treatment. Present study suggested that EYP treatment enhanced osteogenic activities and increased bone matrix osteogenicgenes. These results could provide a mechanistic explanation for the bone-strengthening effects of EYP.

Keywords: egg yolk peptide, osteoblastic MG-63 cells, alkaline phosphatase, collagen synthesis, osteogenic genes, COL1A1, osteocalcin, osteopontin

Procedia PDF Downloads 368

Authors: Magdalena Rost-Roszkowska, Izabela Poprawa, Alina Chachulska-Zymelka, Lukasz Chajec, Grazyna Wilczek, Piotr Wilczek, Malgorzata Lesniewska

Abstract:

Lithobius forficatus, commonly known as the brown centipede, is a widespread European species, which lives in the upper layers of soil, under stones, litter, rocks, and leaves. As the soil organism, it is exposed to numerous stressors such as xenobiotics, including heavy metals, temperature, starvation, pathogens, etc. Heavy metals are treated as the environmental pollutants of the soil because of their toxic effects on plants, animals and human being. One of the heavy metals which is xenobiotic and can be taken up by plants or animals from the soil is cadmium. The digestive system of centipedes is composed of three distinct regions: fore-, mid- and hindgut. The salivary glands of centipedes are the organs which belong to the anterior region of the digestive system and take part in the synthesis, accumulation, and secretion of many substances. The middle region having contact with the food masses is treated as one of the barriers which protect the organism against any stressors which originate from the external environment, e.g., toxic metals. As the material for our studies, we chose two organs of the digestive system in brown centipede, the organs which take part in homeostasis maintenance: the salivary glands and the midgut. The main purpose of the project was to investigate the relationship between the percentage of depolarized mitochondria, mitophagy and ATP level in cells of mentioned above organs. The animals were divided into experimental groups: K – the control group, the animals cultured in a laboratory conditions in a horticultural soil and fed with Acheta domesticus larvae; Cd1 – the animals cultured in a horticultural soil supplemented with 80 mg/kg (dry weight) of CdCl2, fed with A. domesticus larvae maintained in tap water, 12 days – short-term exposure; Cd2 – the animals cultured in a horticultural soil supplemented with 80 mg/kg (dry weight) of CdCl2, fed with A. domesticus larvae maintained in tap water, 45 days – long-term exposure. The studies were conducted using transmission electron microscopy (TEM), flow cytometry and confocal microscopy. Quantitative analysis revealed that regardless of the organ, a progressive increase in the percentage of cells with depolarized mitochondria was registered, but only in the salivary glands. These were statistically significant changes from the control. In both organs, there were no differences in the level of the analyzed parameter depending on the duration of exposure of individuals to cadmium. Changes in the ultrastructure of mitochondria have been observed. With the extension of the body's exposure time to metal, an increase in the ADP/ATP index was recorded. However, changes statistically significant to the control were demonstrated in the intestine and salivary glands. The size of this intestinal index and salivary glands in the Cd2 group was about thirty and twenty times higher, respectively than in control. Acknowledgment: The study has been financed by the National Science Centre, Poland, grant no 2017/25/B/NZ4/00420.

Keywords: cadmium, digestive system, ultrastructure, centipede

Procedia PDF Downloads 111

Authors: Josephine Hai, Jie Jiang, Karen M. Lyons

Abstract:

Of the musculoskeletal tissues, tendon is least understood in terms of biological development. The current study examines Cysteine-rich angiogenic inducer 61, or CCN1, a member of the CCN family of secreted matricellular proteins that regulate cell behavior via intercellular signaling. Though CCN1 is notable in limiting fibrosis by inducing senescence in fibroblasts, little is known about its role in normal fibrous tissue, where it may be essential to the development of ECM-rich structures like tendons. We found that CCN1 knockout mice (using limb-specific Prx1-Cre) exhibited clubfoot and waddling gaits, a unique phenotype not described in any other mutant to date. Histological analysis showed that the Achilles and patellar tendons, where we previously found high CCN1 expression in adult reporter mice, were thicker and denser in the Prx1-Cre knockouts than in their wildtype littermates. We then hypothesized that CCN1 is required directly in tendon progenitor cells for normal tendon development and generated tendon-specific CCN1 knockout mice using Scx-Cre. We observed similar Achilles/patellar tendon morphology among the Scx-Cre and Prx1-Cre mutants, indicating that the phenotype is a direct result of CCN1’s loss in tendon. To further study phenotype onset and progression, we will histologically characterize these tendons across different developmental time-points. We will also perform RNA-seq and qPCR to analyze tenocyte gene expression and expect fibrotic marker upregulation in the Scx-Cre mutants if CCN1 is required to maintain a normal tendon phenotype. Thus, our study aims to elucidate the molecular mechanisms underlying tendon formation and maintenance. Understanding tendons at the most basic level invites novel approaches to tendon repair.

Keywords: development, matricellular, musculoskeletal, tendon

Procedia PDF Downloads 152

Authors: Masoud Soltanialvar, Ali Bagherpour

Abstract:

Documented cases of human infection with H9N2 avian influenza viruses, first detected in 1999 in Hong Kong and China, indicate that these viruses can be directly transmitted from birds to humans. In this study, we characterized the mutation in the Hemagglutinin (HA) genes and proteins that correlates with a shift in affinity of the Hemagglutinin (HA) protein from the “avian” type sialic receptors to the “human” type in 10 Iranian isolates. We delineated the genomes and receptor binding profile of HA gene of some field isolates and established their phylogenetic relationship to the other Asian H9N2 sub lineages. A total of 1200 tissue samples collected from 40 farms located in various states of Iran during 2008 – 2010 as part of a program to monitor Avian Influenza Viruses (AIV) infection. To determine the genetic relationship of Iranian viruses, the Hemagglutinin (HA) genes from ten isolates were amplified and sequenced (by RT-PCR method). Nucleotide sequences (orf) of the (HA) genes were used for phylogenetic tree construction. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) in all ten Iranian isolates which indicates a preference to binding of α (2–6) sialic acid receptors, so these Iranian H9N2 viruses have the potential to infect human beings. These isolates showed high degree of homology with 2 human H9N2 isolates A/HK/1073/99, A/HK/1074/99. Phylogenetic analysis of showed that all the HA genes of the Iranian H9N2 viruses fall into a single group within a G1-like sublineage which had contributed as donor of six internal genes to H5N1 highly pathogenic avian influenza. The results of this study indicated that all Iranian viruses have the potential to emerge as highly pathogenic influenza virus, and considering the homology of these isolates with human H9N2 strains, it seems that the potential of these avian influenza isolates to infect human should not be overlooked.

Keywords: influenza virus, hemagglutinin, neuraminidase, Iran

Procedia PDF Downloads 425

Authors: Andrey V. Khromov, Antonida V. Makhotenko, Ekaterina A. Snigir, Svetlana S. Makarova, Natalia O. Kalinina, Valentin V. Makarov, Mikhail E. Taliansky

Abstract:

Due to its simplicity, CRISPR/Cas9 has become widely used and capable of inducing mutations in the genes of organisms of various kingdoms. The aim of this work was to develop applications for the efficient modification of DNA coding sequences of phytoene desaturase (PDS), coilin and vacuolar invertase (Solanum tuberosum) genes, and to develop a new nanoparticles carrier efficient technology to deliver the CRISPR/Cas9 system for editing the plant genome. For each of the genes - coilin, PDS and vacuolar invertase, five single RNA guide (sgRNAs) were synthesized. To determine the most suitable nanoplatform, two types of NP platforms were used: magnetic NPs (MNPS) and gold NPs (AuNPs). To test the penetration efficiency, they were functionalized with fluorescent agents - BSA * FITS and GFP, as well as labeled Cy3 small-sized RNA. To measure the efficiency, a fluorescence and confocal microscopy were used. It was shown that the best of these options were AuNP - both in the case of proteins and in the case of RNA. The next step was to check the possibility of delivering components of the CRISPR/Cas9 system to plant cells for editing target genes. AuNPs were functionalized with a ribonucleoprotein complex consisting of Cas9 and corresponding to target genes sgRNAs, and they were biolistically bombarded to axillary buds and apical meristems of potato plants. After the treatment by the best NP carrier, potato meristems were grown to adult plants. DNA isolated from this plants was sent to a preliminary fragment of the analysis to screen out the non-transformed samples, and then to the NGS. The present work was carried out with the financial support from the Russian Science Foundation (grant No. 16-16-04019).

Keywords: biobombardment, coilin, CRISPR/Cas9, nanoparticles, NPs, PDS, sgRNA, vacuolar invertase

Procedia PDF Downloads 289

Authors: Samaneh Nazemi, Ayla Bahramian, Marzieh Aghazadeh

Abstract:

Saliva is a clear liquid composed of water, electrolytes, glucose, amylase, glycoproteins, and antimicrobial enzymes. The presence of a wide range of molecules and proteins in saliva has made this fluid valuable in screening for some diseases as well as epidemiological studies. Saliva is easier than serum to collect in large populations. Due to the importance of sodium and glucose levels in many biological processes, this study investigates the relationship between sodium and glucose levels in salivary and serum samples of healthy individuals referring to Tabriz Dental School. This descriptive-analytical study was performed on 40 healthy individuals referred to the Oral Diseases Department of Tabriz Dental School. Serum and saliva samples were taken from these patients according to standard protocols. Data were presented as mean (standard deviation) and frequency (percentage) for quantitative and qualitative variables. Pearson test, paired-samples T-test and SPSS 24 software were used to determine the correlation between serum and salivary levels of these biomarkers. In this study, P less than 0.05% is considered significant. Out of 40 participants in this study, 14 (35%) were male, and 26 (65%) were female. According to the results of this study, the mean salivary sodium (127.53 ml/dl) was lower than the mean serum sodium (141.2725 ml/dl). In contrast, the mean salivary glucose (4.55 ml/dl) was lower than the mean serum glucose (89.7575 ml/dl). The result of paired samples T-test (p-value<0.05) showed that there is a statistically significant difference between the mean of serum sodium and salivary sodium, as well as between the serum glucose and salivary glucose. Pearson correlation test results showed that there is no significant correlation between serum sodium and salivary sodium (p-value >0.05), but here is a positive correlation between serum glucose and salivary glucose (p-value<0.001). Both serum sodium and glucose were higher than salivary sodium and glucose.In conclusion, this study found that there was not a statistical relationship between salivary glucose and serum glucose and also salivary sodium and serum sodium of healthy individuals. Perhaps salivary samples can’t be used to measure glucose and sodium in these individuals.

Keywords: glucose, saliva, serum, sodium

Procedia PDF Downloads 228

Authors: Ife O. Bolaji

Abstract:

Environmental concerns arising from the use of fossil fuels has increased the interest in the development of renewable and sustainable sources of energy. This would minimize the dependence on fossil fuels and serve as future alternatives. Organic wastes contain carbohydrates, proteins and lipids, which can be utilised as carbon sources for the production of bio-based products. Cellulose is the most abundant natural biopolymer, being the main structural component of lignocellulosic materials. The aim of this project is to develop a biological process for the hydrolysis and fermentation of organic wastes into ethanol and organic acids. The hydrolysis and fermentation processes are integrated in a single vessel using undefined mixed culture microorganisms. The anaerobic fermentation of microcrystalline cellulose was investigated in continuous and batch reactors at 25°C with an appropriate growth medium for cellulase formation, hydrolysis, and fermentation. The reactors were inoculated with soil (B1, C1, C3) or sludge from an anaerobic digester (B2, C2) and the breakdown of cellulose was monitored by measuring the production of ethanol, organic acids and the residual cellulose. The batch reactors B1 and B2 showed negligible microbial activity due to inhibition while the continuous reactors, C1, C2 and C3, exhibited little cellulose hydrolysis which was concealed by the cellulose accumulation in the reactor. At the end of the continuous operation, the reactors C1, C2 and C3 were operated under batch conditions. 48%, 34% and 42% cellulose had been fermented by day 88, 55 and 55 respectively of the batch fermentation. Acetic acid, ethanol, propionic acid and butyric acids were the main fermentation products in the reactors. A stable concentration of 0.6 g/l ethanol and 5 g/L acetic acid was maintained in C3 for several weeks due to reduced activity of methanogens caused by the decrease in pH. Thus far, the results have demonstrated that mixed microbial culture is capable of hydrolysing and fermenting cellulose under lenient conditions. The fermentation of cellulose has been found effective in a combination of continuous and batch processes.

Keywords: cellulose, hydrolysis, mixed culture, organic waste

Procedia PDF Downloads 343

Authors: Hongquan Zhang

Abstract:

Inspired by endogenous protein motors, researchers have constructed various synthetic DNA motors based on the specificity and predictability of Watson-Crick base pairing. However, the application of DNA motors to signal amplification and biosensing is limited because of low mobility and difficulty in real-time monitoring of the walking process. The objective of our work was to construct a new type of DNA motor termed target-triggered DNA motors that can walk for hundreds of steps in response to a single target binding event. To improve the mobility and processivity of DNA motors, we used gold nanoparticles (AuNPs) as scaffolds to build high-density, three-dimensional tracks. Hundreds of track strands are conjugated to a single AuNP. To enable DNA motors to respond to specific protein and nucleic acid targets, we adapted the binding-induced DNA assembly into the design of the target-triggered DNA motors. In response to the binding of specific target molecules, DNA motors are activated to autonomously walk along AuNP, which is powered by a nicking endonuclease or DNAzyme-catalyzed cleavage of track strands. Each moving step restores the fluorescence of a dye molecule, enabling monitoring of the operation of DNA motors in real time. The motors can translate a single binding event into the generation of hundreds of oligonucleotides from a single nanoparticle. The motors have been applied to amplify the detection of proteins and nucleic acids in test tubes and live cells. The motors were able to detect low pM concentrations of specific protein and nucleic acid targets in homogeneous solutions without the need for separation. Target-triggered DNA motors are significant for broadening applications of DNA motors to molecular sensing, cell imagining, molecular interaction monitoring, and controlled delivery and release of therapeutics.

Keywords: biosensing, DNA motors, gold nanoparticles, signal amplification

Procedia PDF Downloads 60

Authors: F. Bellali, M. Kharroubi, Y. Rady, N. Bourhim

Abstract:

In Morocco, fish processing industry is an important source income for a large amount of by-products including skins, bones, heads, guts, and scales. Those underutilized resources particularly scales contain a large amount of proteins and calcium. Sardina plichardus scales from resulting from the transformation operation have the potential to be used as raw material for the collagen production. Taking into account this strong expectation of the regional fish industry, scales sardine upgrading is well justified. In addition, political and societal demands for sustainability and environment-friendly industrial production systems, coupled with the depletion of fish resources, drive this trend forward. Therefore, fish scale used as a potential source to isolate collagen has a wide large of applications in food, cosmetic, and biomedical industry. The main aim of this study is to isolate and characterize the acid solubilize collagen from sardine fish scale, Sardina pilchardus. Experimental design methodology was adopted in collagen processing for extracting optimization. The first stage of this work is to investigate the optimization conditions of the sardine scale deproteinization on using response surface methodology (RSM). The second part focus on the demineralization with HCl solution or EDTA. And the last one is to establish the optimum condition for the isolation of collagen from fish scale by solvent extraction. The advancement from lab scale to pilot scale is a critical stage in the technological development. In this study, the optimal condition for the deproteinization which was validated at laboratory scale was employed in the pilot scale procedure. The deproteinization of fish scale was then demonstrated on a pilot scale (2Kg scales, 20l NaOH), resulting in protein content (0,2mg/ml) and hydroxyproline content (2,11mg/l). These results indicated that the pilot-scale showed similar performances to those of lab-scale one.

Keywords: deproteinization, pilot scale, scale, sardine pilchardus

Procedia PDF Downloads 421

Authors: Yeon Ho Je

Abstract:

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an_65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

Keywords: baculovirus, insecticide, neurotoxin, neurobactrus

Procedia PDF Downloads 292

Authors: Basanti Ekka, Talis Juhna

Abstract:

Dairy industrial effluents originated by the typical processing activities are composed of various organic and inorganic constituents, and these include proteins, fats, inorganic salts, antibiotics, detergents, sanitizers, pathogenic viruses, bacteria, etc. These contaminants are harmful to not only human beings but also aquatic flora and fauna. Because consisting of large classes of contaminants, the specific targeted removal methods available in the literature are not viable solutions on the industrial scale. Therefore, in this on-going research, a series of coagulation, electrochemical, and catalytic methods will be employed. The bulk coagulation and electrochemical methods can wash off most of the contaminants, but some of the harmful chemicals may slip in; therefore, specific catalysts designed and synthesized will be employed for the removal of targeted chemicals. In the context of Latvian dairy industries, presently, work is under progress on the characterization of dairy effluents by total organic carbon (TOC), Inductively Coupled Plasma Mass Spectrometry (ICP-MS)/ Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), High-Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS), and Mass Spectrometry. After careful evaluation of the dairy effluents, a cost-effective natural coagulant will be employed prior to advanced electrochemical technology such as electrocoagulation and electro-oxidation as a secondary treatment process. Finally, graphene oxide (GO) based hybrid materials will be used for post-treatment of dairy wastewater as graphene oxide has been widely applied in various fields such as environmental remediation and energy production due to the presence of various oxygen-containing groups. Modified GO will be used as a catalyst for the removal of remaining contaminants after the electrochemical process.

Keywords: catalysis, dairy wastewater, electrochemical method, graphene oxide

Procedia PDF Downloads 113

Authors: S. Essa, H. Safar, R. Raghupathy

Abstract:

Human cytomegalovirus (CMV) continues to be a source of severe complications to immunologically immature and immune-compromised hosts. Effective CMV vaccine that diminishes CMV disease in transplant patients and avoids congenital infection remains of high importance as no approved vaccines exist. Though the exact links of defense mechanisms are unidentified, viral-specific antibodies and Th1/Th2 cytokine responses have been involved in controlling viral infections. CMV envelope glycoprotein B (UL55/gB), the matrix proteins (UL83/pp65, UL99/pp28, UL32/pp150), and the assembly protein UL80a/pp38 are known to be targets of antiviral immune responses. In this study, mice were immunized with five HCMV antigens (UL32/pp150, UL80a/pp38, UL99/pp28, and UL83/pp65), and serum samples were collected and evaluated for eliciting viral-specific antibody responses. Moreover, Splenocytes were collected, stimulated, and assessed for cytokine responses. The results demonstrated a CMV-antigen-specific antibody response to pp38 and pp65 (E/C >2.0). The highest titers were detected with pp38 (average E/C 16.275) followed by pp65 (average E/C 7.72). Compared to control cells, splenocytes from PP38 antigen immunized mice gave a significantly higher concentration of GM-CSF, IFN-γ, IL-2 IL-4, IL-5, and IL-17A (P<0.05). Also, splenocytes from pp65 antigen immunized mice resulted in a significantly higher concentration of GM-CSF, IFN-γ, IL-2 IL-4, IL-10, IL-12, IL-17A, and TNF- α. The designation of target CMV peptides by identifying viral-specific antibodies and cytokine responses is vital for understanding the protective immune mechanisms during CMV infection and identifying appropriate viral antigens to develop novel vaccines.

Keywords: hepatitis C virus, peripheral blood mononuclear cells, neutrophils, cytokines

Procedia PDF Downloads 108

Authors: Magdalena B. Kaziuk, Waldemar Kosiba

Abstract:

Background: In the XXI century we have to deal with the epidemic of obesity which is important risk factor for the cardiovascular and kidney diseases. Lipo proteins are directly involved in the atherosclerotic process. Non-high-density lipo protein (non-HDL) began following widespread recognition of its superiority over LDL as a measurement of vascular event risk. Non-HDL includes residual risk which persists in patients after achieved recommended level of LDL. Materials and Methods: The study covered 111 patients (52 females, 59 males, age 51,91±14 years), hospitalized on the intern department. Body composition was assessed using the bioimpendance method and anthropometric measurements. Physical activity data were collected during the interview. The nutritional status and the obesity type were determined with the Waist to Height Ratio and the Waist to Hip Ratio. A function of the kidney was evaluated by calculating the estimated glomerular filtration rate (eGFR) using MDRD formula. Non-HDL was calculated as a difference between concentration of the Total and HDL cholesterol. Results: 10% of patients were found to be underweight; 23.9 % had correct body weight; 15,08 % had overweight, while the remaining group had obesity: 51,02 %. People with the android shape have higher non-HDL cholesterol versus with the gynoid shape (p=0.003). The higher was non-HDL, the lower eGFR had studied subjects (p < 0.001). Significant correlation was found between high non-HDL and incorrect dietary habits in patients avoiding eating vegetables, fruits and having low physical activity (p < 0.005). Conclusions: Android type of figure raises the residual risk of the heart disease associated with higher levels of non-HDL. Increasing physical activity in these patients reduces the level of non-HDL. Non-HDL seems to be the best predictor among all cholesterol measures for the cardiovascular events and worsening eGFR.

Keywords: obesity, non-HDL cholesterol, glomerular filtration rate, lifestyle

Procedia PDF Downloads 356

Authors: Hai-Ying Liu, Yi-Hao Chen, Shu-Yin Pang, Feng-Hua Wang, Xiao-Fang Peng, Li-Yuan Yang, Zheng-Rong Chen, Yi Chen, Bing Zhu

Abstract:

Objective: Biliary atresia (BA) is characterized by progressive liver fibrosis. Epithelial-mesenchymal transition (EMT) has been implicated as a key mechanism in the pathogenesis of organ fibrosis. MiR-200a has been shown to repress EMT. We aim to explore the role of miR-200a in the fibrogenesis of BA. Methods: We obtained the plasma samples and liver samples from patients with BA or controls to examine the role of miR-200a. Histological liver fibrosis was assessed using the Ishak fibrosis scores. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of miR-200a in plasma. We also evaluated the expression of miR-200a in liver tissues using tyramide signal amplification fluorescence in situ hybridization (TSA-FISH). The expression of EMT related proteins zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin and α-smooth muscle actin (α-SMA) in the liver sections were detected by immunohistochemical staining. Results: We found that the expression of miR-200a was both elevated in the plasma and liver tissues from BA patients compared with the controls. The hepatic expression of ZEB1 and α-SMA were markedly increased in the liver sections from BA patients compared to the controls, whereas E-cadherin was downregulated in the BA group. Simultaneously, we noted that the hepatic expression of miR-200a, E-cadherin and α-SMA were upregulated with the progression of liver fibrosis in the BA group, while ZEB1 was downregulated with the progression of liver fibrosis in BA patients. Conclusion: These findings suggest EMT has a critical effect on the fibrotic process of BA, and the interaction between miR-200a and ZEB1 may regulate EMT and eventually influence liver fibrogenesis of BA.

Keywords: biliary atresia, liver fibrosis, MicroRNA, epithelial-mesenchymal transition, zinc finger E-box-binding homeobox 1

Procedia PDF Downloads 337

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

Abstract:

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 299

Authors: Sofia G. Meirinho, Luis G. Dias, António M. Peres, Lígia R. Rodrigues

Abstract:

The emerging development of electrochemical aptasen sors has enabled the easy and fast detection of protein biomarkers in standard and real samples. Biomarkers are produced by body organs or tumours and provide a measure of antigens on cell surfaces. When detected in high amounts in blood, they can be suggestive of tumour activity. These biomarkers are more often used to evaluate treatment effects or to assess the potential for metastatic disease in patients with established disease. Osteopontin (OPN) is a protein found in all body fluids and constitutes a possible biomarker because its overexpression has been related with breast cancer evolution and metastasis. Currently, biomarkers are commonly used for the development of diagnostic methods, allowing the detection of the disease in its initial stages. A previously described RNA aptamer was used in the current work to develop a simple and sensitive electrochemical aptasensor with high affinity for human OPN. The RNA aptamer was biotinylated and immobilized on a gold electrode by avidin-biotin interaction. The electrochemical signal generated from the aptamer–target molecule interaction was monitored electrochemically using cyclic voltammetry in the presence of [Fe (CN) 6]−3/− as a redox probe. The signal observed showed a current decrease due to the binding of OPN. The preliminary results showed that this aptasensor enables the detection of OPN in standard solutions, showing good selectivity towards the target in the presence of others interfering proteins such as bovine OPN and bovine serum albumin. The results gathered in the current work suggest that the proposed electrochemical aptasensor is a simple and sensitive detection tool for human OPN and so, may have future applications in cancer disease monitoring.

Keywords: osteopontin, aptamer, aptasensor, screen-printed electrode, cyclic voltammetry

Procedia PDF Downloads 406

Authors: Thanusha D. Abeywickrama, Inoka C. Perera, Genji Kurisu

Abstract:

Tuberculosis, considered being as the ninth leading cause of death worldwide, cause from a single infectious agent M. tuberculosis and the drug resistance nature of this bacterium is a continuing threat to the world. Therefore TB preventing treatment is expanding, where this study designed to analyze the regulatory mechanism of GntR transcriptional regulator gene Rv0792c, which lie between several genes codes for some hypothetical proteins, a monooxygenase and an oxidoreductase. The gene encoding Rv0792c was cloned into pET28a and expressed protein was purified to near homogeneity by Nickel affinity chromatography. It was previously reported that the protein binds within the intergenic region (BS region) between Rv0792c gene and monooxygenase (Rv0793). This resulted in binding of three protein molecules with the BS region suggesting tight control of monooxygenase as well as its own gene. Since monooxygenase plays a key role in metabolism, this gene may have a global regulatory role. The natural ligand for this regulator is still under investigation. In relation to the Rv0792 protein structure, a Circular Dichroism (CD) spectrum was carried out to determine its secondary structure elements. Percentage-wise, 17.4% Helix, 21.8% Antiparallel, 5.1% Parallel, 12.3% turn and 43.5% other were revealed from CD spectrum data under room temperature. Differential Scanning Calorimetry (DSC) was conducted to assess the thermal stability of Rv0792, which the melting temperature of protein is 57.2 ± 0.6 °C. The graph of heat capacity (Cp) versus temperature for the best fit was obtained for non-two-state model, which concludes the folding of Rv0792 protein occurs through stable intermediates. Peak area (∆HCal ) and Peak shape (∆HVant ) was calculated from the graph and ∆HCal / ∆HVant was close to 0.5, suggesting dimeric nature of the protein.

Keywords: CD spectrum, DSC analysis, GntR transcriptional regulator, protein structure

Procedia PDF Downloads 203

Authors: Kai Pei Huang, Ting Chung Hung, Li Ching Wu

Abstract:

Amyloidosis refers to a variety of conditions wherein amyloid proteins are abnormally deposited in organ or tissues and cause harm. Among the several forms of amyloidosis, the principal types of that in inpatient medical services are the AL amyloidosis (primary) and AA amyloidois (secondary). AL Amyloidois is due to deposition of protein derived from overproduction of immunoglobulin light chain in plasma cell myeloma. Furthermore, it is a systemic disorder that can present with a variety of symptoms, including heavy proteinemia and edema, heptosplenomegaly, otherwise unexplained heart failure. We reported a 78-year-old female presenting dysuria, oliguria and leg edema for several months. Laboratory data showed proteinuria (UPCR:1679.8), leukocytosis (WBC:16.2 x 10^3/uL), results of serum urea nitrogen (39mg/dL), creatinine (0.76 mg/dL), IgG (748 mg/dL.), IgA (635 mg/dL), IgM (63 mg/dL), kappa light chain(18.8 mg/dL), lambda light chain (110.0 mg/dL) and kappa/lambda ratio (0.17). Renal biopsy found amyloid fibrils in glomerular mesangial area, and Congo red stain highlights amyloid deposition in glomeruli. Additional lab studies included serum protein electrophoresis, which shows a major monoclonal peak in β region and minor small peak in gamma region, and the immunotyping studies for serum showed two IgA/λ type. We treated sample with beta-mercaptoethanol which reducing the polymerized immunoglobulin to clarify two IgA/λ are secreted from the same plasma cell clone in bone marrow. Later examination confirmed it existed plasma cell infiltration in bone marrow, and the immunohistochemical staining showed monotypic for λ light chain and are positive for IgA. All findings mentioned above reveal it is a case of plasma cell myeloma with λ Light Chain Amyloidosis.

Keywords: amyloidosis, immunoglobulin light chain, plasma cell myeloma, serum protein electrophoresis

Procedia PDF Downloads 193

Authors: Ouchemoukh Salim, Amessis-Ouchemoukh Nadia, Guenaoui Nawel, Moumeni Lynda, Zaidi Hicham, Otmani Amar, Sadou Dyhia

Abstract:

Honey is a hive food rich in carbohydrates and water and it also has a lot of nutrients (enzymes, minerals, organic acids, phytochemicals...). It is used in different nutritional and therapeutic fields. Algerian honey was studied for its physicochemical parameters, nutritional values (moisture, brix, pH, electrical conductivity, and amounts of HMF, proteins, proline, total phenolic compounds and flavonoids) and some biological activities (antioxidant, anti-inflammatory and enzymatic anti-browning). The antioxidant activities of the samples were estimated using different methods (ABTS, DPPH free radicals scavenging, reducing power, and chelating ferrous activity). All honeys were acidic (3.45≤pH≤4.65). The color varied from mimosa yellow to dark brown. The specific rotation was levorotatory in most honey samples, and the electrical conductivity, hydroxymethylfurfural, and proline values agreed with the international honey requirements. For anti-inflammatory activity, the results showed that the inhibiting capacity of the denaturation of the BSA of the honey analyzed varied from 15 to 75 % with a maximum of activity at the concentration of 0,5 mg/ml. All honey exhibited enzymatic anti-browning on different slices of fruits. In fact, the results showed that the controls have the greatest browning unit compared to the honeys studied and PPO and POD enzymes had the lowest enzyme activity. High significant correlations were found between the color of honey, its antioxidant content and its biological activities (antioxidant, anti-inflammatory and enzymatic anti-browning). The dark color of honey is a good indicator of the best biological properties, therefore, the best nutritional and therapeutic values.

Keywords: honey, physico-chemical parameters, bioactive compounds, biological properties

Procedia PDF Downloads 25

Authors: Jasna Čanadanović-Brunet, Gordana Ćetković, Sonja Djilas, Vesna Tumbas-Šaponjac, Jelena Vulić, Sladjana Stajčić

Abstract:

Honey, produced by the honeybee, is a natural saturated sugar solution, which is mainly composed of a complex mixture of carbohydrates. Besides this, it also contains certain minor constituents, proteins, enzymes, amino and organic acids, lipids, vitamins, phenolic acids, flavonoids and carotenoids. Honey serves as a source of natural antioxidants, which are effective in reducing the risk of heart disease, cancer, immune-system decline, cataracts, and different inflammatory processes. Honey is consumed in its natural form alone, but also in combination with nuts and various kinds of dried fruits (plums, figs, cranberries, apricots etc.). The aim of this research was to investigate the contribution of dried apricot addition to polyphenols and flavonoids contents and antioxidant activities of honey. Some individual phenolic compounds in Serbian polyfloral honey (PH), linden honey (LH) and also in their mixtures with dried apricot, in 40% mass concentrations (PH40; LH40), were identified and quantified by HPLC. The most dominant phenolic compound was: gallic acid in LH (11.14 mg/100g), LH40 (42.65 mg/100g), PH (7.24 mg/100g) and catehin in PH40 (11.83 mg/100g). The antioxidant activity of PH, LH, PH40 and LH40 was tested by measuring their ability to scavenge hydroxyl radicals (OH) by electron spin resonance spectroscopy (ESR). Honey samples with 40% dried apricot exhibited better antioxidant activity measured by hydroxyl radical scavenging activity. The EC50 values, the amount of antioxidant necessary to decrease the initial concentration of OH radicals by 50%, were: EC50PH=3.36 mg/ml, EC50LH=13.36 mg/ml, EC50PH40=2.29 mg/ml, EC50 LH40=7.78 mg/ml. Our results indicate that supplementation of polyfloral honey and linden honey with dried apricots improves antioxidant activity of honey by enriching the phenolic composition.

Keywords: honey, dried apricot, HPLC, hydroxyl radical

Procedia PDF Downloads 333

Authors: Ivan Zupan, Tomislav Saric, Suzana Tkalcic

Abstract:

IMUNO-2865® is a commercially available, β–glucan based, natural hemicellulose compound with proven immunostimulative properties in people, domestic and some aquatic animals. During the experimental feeding trial with IMUNO-2865® in juvenile wild-caught chub under laboratory conditions, supplementation resulted in overall higher growth performance for all experimental groups regardless of the concentration of the added compound. The maximum, 5% concentration of the supplement, resulted in highest weight gain and calculated specific growth rate. In sea bream, as economically most important species in the Mediterranean aquaculture, significant increases in numbers of monocytes and heterophils were observed in the group supplemented with 2.5 % of IMUNO-2865® in the feed. An overall increase of erythrocytes was noted by the end of the experiment, although with variable distribution among groups. Blood Ca++ levels, total proteins, and total NH₃ were significantly higher after 60 days of feeding in all treatment groups compared to the control and remained elevated in the treated group following the secession of supplementation. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and serum paraoxonase PON1 (U/L) showed similar trends. All these parameters are playing a significant role in either oxygen supplementation of tissues, or anabolic and catabolic processes that on molecular levels contribute to the overall health and immune-building capacity of cells and tissues. The complete lack of mortality in sea bream and presented increases in cellular, biochemical and oxidative stress parameters in the blood suggest that the IMUNO-2865® represents a safe dietary supplement for in aquaculture, with an overall positive and potentially immunostimulative effect on farmed fish.

Keywords: IMUNO-2865®, β–glucans, Mediterranean aquaculture, fish imunnostimulans

Procedia PDF Downloads 119

Authors: Boudjenah-Haroun Saliha, Isselnane Souad, Nouani Abdelwahab, Baaissa Babelhadj, Mati Abderrahmane

Abstract:

Algeria is experiencing significant development of the dairy sector, where consumption of milk and milk products increased by 2.7 million tons in 2008 to 4,400,000 tons in 2013, and cheese production has reached 1640 tons in the year 2014 with average consumption of 0.7 kg/person/year. Although rennet is still the most used coagulating enzyme in cheese, its production has been growing worldwide shortage. This shortage is primarily due to a growing increase in the production and consumption of cheese, and the inability to increase in parallel the production of rennet. This shortage has caused very large fluctuations in its price). To overcome these obstacles, much research has been undertaken to find effective and competitive substitutes used industrially. For this, the selection of a local production of rennet substitute is desirable. It would allow a permanent supply with limited dependence on imports and price fluctuations. Investigations conducted by our research team showed that extracts coagulants from the stomachs of older camels are characterized by a coagulating power than those from younger camels. The objective of this work is to study the possibility of substituting commercial rennet coagulant by gastric enzymes from adult camels for coagulation bovine milk. Excerpts from the raw camel coagulants obtained are characterized through their teneures proteins and clotting and proteolytic activities. Milk clotting conditions by the action of these extracts were optimized. Milk clotting time all treated with enzyme preparations and under different conditions was calculated. Bovine rennet has been used for comparison. The results show that crude extracts from gastric adult camel can be good substituting bovine rennet.

Keywords: Algeria, camel, cheese, coagulation, gastric extracts, milk

Procedia PDF Downloads 411

Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

Abstract:

Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

Procedia PDF Downloads 234

Authors: Szu-Chieh Yu, Pei-Chin Chiand, Chih-Yi Lin, Yi-Wen Chien

Abstract:

UV radiation from sunlight cause numbers of acute and chronic skin damage which can result in inflammation, immune changes, physical changes and DNA damage that facilitates skin aging and the development of skin carcinogenesis. Reactive oxygen species (ROS) are generated by excessive solar UV radiation, resulting in oxidative damage to cellar components, proteins, lipids, and nucleic acids. Thus, antioxidation plays an important role that protects skin against ROS-induced injury. Safflower (Carthamus tinctorius L.) is an important Chinese medicine contained abundance flavones and hydroxysafflor yellow A (HSYA) which is main active ingredient. HSYA is part of quinochalcone and has unique structures of hydroxy groups that provided the antioxidant effect. In this study, the aim was to investigate the protective role of HYSA in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism. The HaCaT cells were UVA-irradiated and the effects of HYSA on cell viability, reactive oxygen species generation, DNA fragmentation and lipid peroxidation were measured. The mRNA expression of matrix metalloproteinase Ι (MMP Ι), cyclooxygenase-2 (COX-2) were determined by RT-PCR. In this study, UVA exposure lead to decrease in cell viability and increase in reactive oxygen species generation in HaCaT cells. HYSA could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, HYSA can reduce inflammation through inhibition the mRNA expression of MMP Ι and COX-2. Our results suggest that HSYA can act as a free radical scavenger while keratinocytes were photodamaged. HYSA could be a useful natural medicine for the protection of epidermal cells from UVA-induced damage and will be developed into products for skin care.

Keywords: HaCaT keratinocytes, hydroxysafflor yellow A (HSYA), MMP Ι, oxidative stress

Procedia PDF Downloads 361

Authors: Geno Paul S. Cumla, Jan Mariel M. Gentiles, M. Brenda Gajelan-Samson

Abstract:

Eutrophication is a process where in there is a surplus of nutrients present in a lake or river. Harmful cyanobacteria, hypoxia, and primarily algae, which contain toxins, grow because of the excess nutrients. Algal blooms can cause fish kills, limiting the light penetration which reduces growth of aquatic organisms, causing die-offs of plants and produce conditions that are dangerous to aquatic and human life. The main cause for eutrophication is the presence of excessive amounts of phosphorus (P) and nitrogen (N). Nitrogen is necessary for the production of the plant tissues and is usually used to synthesize proteins. Nitrate is a compound that contains nitrogen, and at elevated levels it can cause harmful effects. Excessive amounts of phosphorus, displaced through human activity, is the major cause of algae growth and as well as degraded water quality. To accomplish this study the Assessment of Soluble inorganic nitrogen (SIN), Assessment of Soluble reactive phosphate (SRP), Determination of Chlorophyll a (Chl-a) concentration, and Determination of Dominating Taxa were done. The study addresses the high probability of algal blooms in Maqueda Bay by assessing the biotic and abiotic factors of Antiao and Jiabong rivers. The data predicts the overgrowth of algae and to create awareness to prevent the event from taking place. The study assesses the adverse effects that could be prevented by understanding and controlling algae. This should predict future cases of algal blooms and allow government agencies which require data to create programs to prevent and assess these issues.

Keywords: eutrophication, chlorophyll a, nitrogen, phosphorus, red tide, Kjeldahl method, spectrophotometer, assessment of soluble inorganic nitrogen, SIN, assessment of soluble reactive phosphate, SRP

Procedia PDF Downloads 117

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

Abstract:

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

Procedia PDF Downloads 111

Authors: Arámbula-Villa Gerónimo, García-Lara Kenia Y., Figueroa-Cárdenas J. D., Pérez-Robles J. F., Jiménez-Sandoval S., Salazar-López R., Herrera-Corredor J. A.

Abstract:

The nixtamalization process (thermal-alkaline method) improves the nutritional part of the corn grain. In this process, the using of Ca(OH)₂ is basic, although the chemical mechanisms between this alkali and the carbohydrates (starch), proteins, lipids, and fiber have not been fully identified. In this study, the native corn starch was taken as a model, and it was subjected to cooking with different concentrations of lime (nixtamalization process) and specific studies of FTIR and XRD were carried out to identify the formation of chemical compounds, and the physical, physicochemical, rheological (paste) and structural properties of material obtained were determined. The FTIR spectra showed the formation of calcium-starch complexes. The treatments with Ca(OH)₂ showed a band shift towards 1675 cm⁻¹ and a band in 1436 cm⁻¹ (COO⁻), indicating the oxidation of starch. Three bands were identified (1575, 1550, and 1540 cm⁻¹) characteristics of carboxylic acid salts for three types of coordinated structures: monodentate, pseudo-bridged, and bidentate. The XRD spectra of starch treated with Ca(OH)₂ showed a peak corresponding to CaCO₃ (29.40°). The oxidation of starch was favored with low concentrations of Ca(OH)₂, producing carboxyl and carbonyl groups and increasing the residual CaCO₃. The increased concentration of Ca(OH)₂ showed the formation of calcium carboxylates, with a decrease in relative crystallinity and residual CaCO₃. Samples with low concentrations of Ca(OH)₂ slowed the onset of gelatinization and increased the swelling of the granules and the peak viscosity. The higher concentrations of Ca(OH)₂ difficulted the water absorption and decreased the viscosity rate and peak viscosity. These results can be used to improve the quality characteristics of the dough and tortillas and to get better acceptance by consumers.

Keywords: maize starch, nixtamalization, gelatinization, calcium carboxylates

Procedia PDF Downloads 68

Authors: K. Nikovics, D. Riccobono, M. Oger, H. Morin, L. Barbier, T. Poyot, X. Holy, A. Bendahmane, M. Drouet, A. L. Favier

Abstract:

Muscle regeneration after injury (as irradiation) is of great importance. However, the molecular and cellular mechanisms are still unclear. Cytokines are believed to play fundamental role in the different stages of muscle regeneration. They are secreted by many cell populations, but the predominant producers are macrophages and helper T cells. On the other hand, it has been shown that adipose tissue derived stromal/stem cell (ASC) injection could improve muscle regeneration. Stem cells probably induce the coordinated modulations of gene expression in different macrophage cells. Therefore, we investigated the patterns and timing of changes in gene expression of different cytokines occurring upon stem cells loading. Muscle regeneration was studied in an irradiated muscle of minipig animal model in presence or absence of ASC treatment (irradiated and treated with ASCs, IRR+ASC; irradiated not-treated with ASCs, IRR; and non-irradiated no-IRR). We characterized macrophage populations by immunolabeling in the different conditions. In our study, we found mostly M2 and a few M1 macrophages in the IRR+ASC samples. However, only few M2b macrophages were noticed in the IRR muscles. In addition, we found intensive fibrosis in the IRR samples. With in situ hybridization and immunolabeling, we analyzed the cytokine expression of the different macrophages and we showed that M2d macrophage are the most abundant in the IRR+ASC samples. By in situ hybridization, strong expression of the transforming growth factor β (TGF-β) was observed in the IRR+ASC but very week in the IRR samples. But when we analyzed TGF-β level with immunolabeling the expression was very different: many M2 macrophages showed week expression in IRR+ASC and few cells expressing stronger level in IRR muscles. Therefore, we investigated the MMP expressions in the different muscles. Our data showed that the M2 macrophages of the IRR+ASC muscle expressed MMP2 proteins. Our working hypothesis is that MMP2 expression of the M2 macrophages can decrease fibrosis in the IRR+ASC muscle by capturing TGF-β.

Keywords: adipose tissue derived stromal/stem cell, cytokine, macrophage, muscle regeneration

Procedia PDF Downloads 213

Authors: Gulyas Erzsebet

Abstract:

The multimerisation of proteins is a common feature of many cellular processes; however, it could also impair protein functions and/or be associated with the occurrence of diseases. Thus, development of a research tool monitoring the appearance/presence of multimeric protein forms has great importance for a variety of research fields. Such a tool is potentially applicable in the ante-mortem diagnosis of certain conformational diseases, such as transmissible spongiform encephalopathies (TSE) and Alzheimer’s disease. These conditions are accompanied by the appearance of aggregated protein multimers, present in low concentrations in various tissues. This detection is particularly relevant for TSE where the handling of tissues derived from affected individuals and of meat products of infected animals have become an enormous health concern. Here we demonstrate the potential of such a multimer detection approach in TSE by developing a facile approach. The Biospiral-Detect system resembles a traditional sandwich ELISA, except that the capturing antibody that is attached to a solid surface and the detecting antibody is directed against the same or overlapping epitopes. As a consequence, the capturing antibody shields the epitope on the captured monomer from reacting with the detecting antibody, therefore monomers are not detected. Thus, MDS is capable of detecting only protein multimers with high specificity. We developed an alternative system as well, where RNA aptamers were employed instead of monoclonal antibodies. In order to minimize degradation, the 3' and 5' ends of the aptamer contained deoxyribonucleotides and phosphorothioate linkages. When compared the monoclonal antibodies-based system with the aptamers-based one, the former proved to be superior. Thus all subsequent experiments were conducted by employing the Biospiral -Detect modified sandwich ELISA kit. Our approach showed an order of magnitude higher sensitivity toward mulimers than monomers suggesting that this approach may become a valuable diagnostic tool for conformational diseases that are accompanied by multimerization.

Keywords: diagnosis, ELISA, Prion, TSE

Procedia PDF Downloads 232

Authors: Gabriela Borilova, Monika Petrakova, Petr Kralik

Abstract:

Nowadays, European producers are increasingly interested in the production of halal meat products. Halal meat has been increasingly appearing in the EU's market network and meat products from European producers are being exported to Islamic countries. Halal criteria are mainly related to the origin of muscle used in production, and also to the way products are obtained and processed. Although the EU has legislatively addressed the question of food authenticity, the circumstances of previous years when products with undeclared horse or poultry meat content appeared on EU markets raised the question of the effectiveness of control mechanisms. Replacement of expensive or not-available types of meat for low-priced meat has been on a global scale for a long time. Likewise, halal products may be contaminated (falsified) by pork or food components obtained from pigs. These components include collagen, offal, pork fat, mechanically separated pork, emulsifier, blood, dried blood, dried blood plasma, gelatin, and others. These substances can influence sensory properties of the meat products - color, aroma, flavor, consistency and texture or they are added for preservation and stabilization. Food manufacturers sometimes access these substances mainly due to their dense availability and low prices. However, the use of these substances is not always declared on the product packaging. Verification of the presence of declared ingredients, including the detection of undeclared ingredients, are among the basic control procedures for determining the authenticity of food. Molecular biology methods, based on DNA analysis, offer rapid and sensitive testing. The PCR method and its modification can be successfully used to identify animal species in single- and multi-ingredient raw and processed foods and qPCR is the first choice for food analysis. Like all PCR-based methods, it is simple to implement and its greatest advantage is the absence of post-PCR visualization by electrophoresis. qPCR allows detection of trace amounts of nucleic acids, and by comparing an unknown sample with a calibration curve, it can also provide information on the absolute quantity of individual components in the sample. Our study addresses a problem that is related to the fact that the molecular biological approach of most of the work associated with the identification and quantification of animal species is based on the construction of specific primers amplifying the selected section of the mitochondrial genome. In addition, the sections amplified in conventional PCR are relatively long (hundreds of bp) and unsuitable for use in qPCR, because in DNA fragmentation, amplification of long target sequences is quite limited. Our study focuses on finding a suitable genomic DNA target and optimizing qPCR to reduce variability and distortion of results, which is necessary for the correct interpretation of quantification results. In halal products, the impact of falsification of meat products by the addition of components derived from pigs is all the greater that it is not just about the economic aspect but above all about the religious and social aspect. This work was supported by the Ministry of Agriculture of the Czech Republic (QJ1530107).

Keywords: food fraud, halal food, pork, qPCR

Procedia PDF Downloads 225