Search results for: adhesion gene

Authors: Muhammad SAFDAR, Mehmet Ozaslan, Musarrat Abbas Khan

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Babesiosis is a haemoparasitic disease due to the multiplication of protozoan’s parasite, Babesia ovis in the red blood cells of the host, and contributes numerous economical losses, including sheep and goat ruminants. The early identification and successful treatment of Babesia Ovis spp. belong to the key steps of control and health management of livestock resources. The objective of this study was to construct a polymerase chain reaction (PCR) based method for the detection of Babesia spp. in small ruminants and to determine the risk factors involved in the spreading of babesiosis infections. A total of 100 blood samples were collected from 50 sheep and 50 goats along with different areas of Muzaffargarh, Pakistan, from randomly selected herds. Data on the characteristics of sheep and goats were collected through questionnaires. Of 100 blood samples examined, 18 were positive for Babesia ovis upon microscopic studies, whereas 11 were positive for the presence of Babesia spp. by PCR assay. For the recognition of parasitic DNA, a set of 500bp oligonucleotide was designed by PCR amplification with sequence 18S rRNA gene for B. ovis. The prevalence of babesiosis in small ruminant’s sheep and goat detected by PCR was significantly higher in female animals (28%) than male herds (08%). PCR analysis of the reference samples showed that the detection limit of the PCR assay was 0.01%. Taken together, all data indicated that this PCR assay was a simple, fast, specific detection method for Babesia ovis species in small ruminants compared to other available methods.

Keywords: Babesia ovis, PCR amplification, 18S rRNA, sheep and goat

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Authors: Reza Talebi

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Wheat (Triticum aestivum) is one of the most important food staples in Iran. Understanding genetic variability among the landrace wheat germplasm is important for breeding. Landraces endemic to Iran are a genetic resource that is distinct from other wheat germplasm. In this study, 60 Iranian landrace wheat accessions were characterized AFLP and ISSR markers. Twelve AFLP primer pairs detected 128 polymorphic bands among the sixty genotypes. The mean polymorphism rate based on AFLP data was 31%; however, a wide polymorphism range among primer pairs was observed (22–40%). Polymorphic information content (PIC value) calculated to assess the informativeness of each marker ranged from 0.28 to 0.4, with a mean of 0.37. According to AFLP molecular data, cluster analysis grouped the genotypes in five distinct clusters. .ISSR markers generated 68 bands (average of 6 bands per primer), which 31 were polymorphic (45%) across the 60 wheat genotypes. Polymorphism information content (PIC) value for ISSR markers was calculated in the range of 0.14 to 0.48 with an average of 0.33. Based on data achieved by ISSR-PCR, cluster analysis grouped the genotypes in three distinct clusters. Both AFLP and ISSR markers able to showed that high level of genetic diversity in Iranian landrace wheat accessions has maintained a relatively constant level of genetic diversity during last years.

Keywords: wheat, genetic diversity, AFLP, ISSR

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Authors: Nishanth Venugopal Menon, Chuah Yon Jin, Samantha Phey, Wu Yingnan, Zhang Ying, Vincent Chan, Kang Yuejun

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Cell Migration is a vital phenomenon that the cells undergo in various physiological processes like wound healing, disease progression, embryogenesis, etc. Cell migration depends primarily on the chemical and physical cues available in the cellular environment. The chemical cue involves the chemokines secreted and gradients generated in the environment while physical cues indicate the impact of matrix properties like nanotopography and stiffness on the cells. Mesenchymal Stem Cells (MSCs) have been shown to have a role wound healing in vivo and its migration to the site of the wound has been shown to have a therapeutic effect. In the field of stem cell based tissue regeneration of bones and cartilage, one approach has been to introduce scaffold laden with MSCs into the site of injury to enable tissue regeneration. In this work, we have studied the combinatorial impact of the substrate physical properties on MSC migration. A microfluidic in vitro model was created to perform the migration studies. The microfluidic model used is a three compartment device consisting of two cell seeding compartments and one migration compartment. Four different PDMS substrates with varying substrate roughness, stiffness and hydrophobicity were created. Its surface roughness and stiffness was measured using Atomic Force Microscopy (AFM) while its hydrphobicity was measured from the water contact angle using an optical tensiometer. These PDMS substrates are sealed to the microfluidic chip following which the MSCs are seeded and the cell migration is studied over the period of a week. Cell migration was quantified using fluorescence imaging of the cytoskeleton (F-actin) to find out the area covered by the cells inside the migration compartment. The impact of adhesion proteins on cell migration was also quantified using a real-time polymerase chain reaction (qRT PCR). These results suggested that the optimal substrate for cell migration would be one with an intermediate level of roughness, stiffness and hydrophobicity. A higher or lower value of these properties affected cell migration negatively. These observations have helped us in understanding that different substrate properties need to be considered in tandem, especially while designing scaffolds for tissue regeneration as cell migration is normally impacted by the combinatorial impact of the matrix. These observations may lead us to scaffold optimization in future tissue regeneration applications.

Keywords: cell migration, microfluidics, in vitro model, stem cell migration, scaffold, substrate properties

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Authors: Chiung-Man Tsai, Chia-Jui Weng

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Leptin (LEP) and leptin receptor (LEPR) both play a crucial role in the mediation of physiological reactions and carcinogenesis and may serve as a candidate biomarker of oral cancer. The present case-control study aimed to examine the effects of single nucleotide polymorphisms (SNPs) of LEP -2548 G/A (rs7799039), LEPR K109R (rs1137100), and LEPR Q223R (rs1137101) with or without interacting to environmental carcinogens on the risk for oral squamous cell carcinoma (OSCC). The SNPs of three genetic allele, from 567 patients with oral cancer and 560 healthy controls in Taiwan were analyzed. All of The three genetic polymorphisms exhibited insignificant (P > .05) effects on the risk to have oral cancer. However, the patients with polymorphic allele of LEP -2548 have a significant low risk for the development of clinical stage (A/G, AOR = 0.670, 95% CI = 0.454–0.988, P < .05; A/G+G/G, AOR = 0.676, 95% CI = 0.467–0.978, P < .05) compared to patients with ancestral homozygous A/A genotype. Additionally, an interesting result was found that the impact of LEP -2548 G/A SNP on oral carcinogenesis in subjects without tobacco consumption (A/G, AOR=2.078, 95% CI: 1.161-3.720, p=0.014; A/G+G/G, AOR=2.002, 95% CI: 1.143-3.505, p=0.015) is higher than subjects with tobacco consumption. These results suggest that the genetic polymorphism of LEP -2548 G/A (rs7799039), LEPR K109R (rs1137100), and LEPR Q223R (rs1137101) were not associated with the susceptibility of oral cancer; SNP in LEP -2548 G/A showed a poor clinicopathological development of oral cancer; Population without tobacco consumption and with polymorphic LEP -2548 G/A gene may significantly increase the risk to have oral cancer.

Keywords: carcinogen, leptin, leptin receptor, oral squamous cell carcinoma, single nucleotide polymorphism

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Authors: Sana Rezig, Yosr Ben Mlik, Mounir Jaouadi, Foued Khoffi, Slah Msahli, Bernard Durand

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Increasing interest in environmental concerns, natural fibers are once again being considered as reinforcements for polymer composites. The main objective of this study is to explore another natural resource, Typha fiber; which is renewable without production cost and available abundantly in nature. The aim of this study was to study the flexural properties of composite resin with and without reinforcing Typha leaf and stem fibers. The specimens were made by the hand-lay-up process using polyester matrix. In our work, we focused on the effect of various treatment conditions (sea water, alkali treatment and a combination of the two treatments), as a surface modifier, on the flexural properties of the Typha fibers reinforced polyester composites. Moreover, weight ratio of Typha leaf or stem fibers was investigated. Besides, both fibers from leaf and stem of Typha plant were used to evaluate the reinforcing effect. Another parameter, which is reinforcement structure, was investigated. In fact, a first composite was made with air-laid nonwoven structure of fibers. A second composite was with a mixture of fibers and resin for each kind of treatment. Results show that alkali treatment and combined process provided better mechanical properties of composites in comparison with fiber treated by sea water. The fiber weight ratio influenced the flexural properties of composites. Indeed, a maximum value of flexural strength of 69.8 and 62,32 MPa with flexural modulus of 6.16 and 6.34 GPawas observed respectively for composite reinforced with leaf and stem fibers for 12.6 % fiber weight ratio. For the different treatments carried out, the treatment using caustic soda, whether alone or after retting seawater, show the best results because it improves adhesion between the polyester matrix and the fibers of reinforcement. SEM photographs were made to ascertain the effects of the surface treatment of the fibers. By varying the structure of the fibers of Typha, the reinforcement used in bulk shows more effective results as that used in the non-woven structure. In addition, flexural strength rises with about (65.32 %) in the case of composite reinforced with a mixture of 12.6% leaf fibers and (27.45 %) in the case of a composite reinforced with a nonwoven structure of 12.6 % of leaf fibers. Thus, to better evaluate the effect of the fiber origin, the reinforcing structure, the processing performed and the reinforcement factor on the performance of composite materials, a statistical study was performed using Minitab. Thus, ANOVA was used, and the patterns of the main effects of these parameters and interaction between them were established. Statistical analysis, the fiber treatment and reinforcement structure seem to be the most significant parameters.

Keywords: flexural properties, fiber treatment, structure and weight ratio, SEM photographs, Typha leaf and stem fibers

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Authors: Anita Singh, Richa Naula, Manoj Raghav

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Nutrient rich and high-yielding varieties of fenugreek can be developed by using genotypes which are naturally high in nutrients. Gene banks harbour scanty germplasm collection of Trigonella spp. and a very little background information about its genetic diversity. The extent of genetic diversity in a specific breeding population depends upon the genotype included in it. The present investigation aims at the estimation of macronutrient (phosphorus by spectrophotometer and potassium by flame photometer), micronutrients, namely, iron, zinc, manganese, and copper from seeds of fenugreek genotypes using atomic absorption spectrophotometer, protein by Rapid N Cube Analyser and Steroidal Saponins. Twenty-eight genotypes of fenugreek along with two standard checks, namely, Pant Ragini and Pusa Early Bunching were collected from different parts of India, and nutrient contents of each genotype were determined at G. B. P. U. A. & T. Laboratory, Pantnagar. Highest potassium content was observed in PFG-35 (1207 mg/100g). PFG-37 and PFG-20 were richest in phosphorus, iron and manganese content among all the genotypes. The lowest zinc content was found in PFG-26 (1.19 mg/100g), while the maximum zinc content was found in PFG- 28 (4.43 mg/100g). The highest content of copper was found in PFG-26 (1.97 mg/100g). PFG-39 has the highest protein content (29.60 %). Significant differences were observed in the steroidal saponin among the genotypes. Saponin content ranged from 0.38 g/100g to 1.31 g/100g. Steroidal Saponins content was found the maximum in PFG-36 (1.31 g/100g) followed by PFG-17 (1.28 g/100g). Therefore, the genotypes which are rich in nutrient and oil content can be used for plant biofortification, dietary supplements, and herbal products.

Keywords: genotypes, macronutrients, micronutrient, protein, seeds

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Authors: Anna Perrone, Federico Martinelli

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Olive fruit is well-known for the high content in secondary metabolites with high interest at nutritional, nutraceutical, antioxidant, and healthy levels. The content of secondary metabolites in olive at harvest may be affected by different water regimes, with significant effects on olive oil composition and quality and, consequently, on its healthy and nutritional features. In this work, a summary of several research studies dealing with the biosynthesis of healthy and nutraceutical metabolites of the secondary metabolism in olive fruit will be reported. The phytochemical findings have been correlated with the expression of key genes involved in polyphenol, terpenoid, and carotenoid biosynthesis and metabolism in response to different development stages and water regimes. Flavonoids were highest in immature fruits, while anthocyanins increased at ripening. In epicarp tissue, this was clearly associated with an up-regulation of the UFGT gene. Olive fruits cultivated under different water regimes were analyzed by metabolomics. This method identified several hundred metabolites in the ripe mesocarp. Among them, 46 were differentially accumulated in the comparison between rain-fed and irrigated conditions. Well-known healthy metabolites were more abundant at a higher level of water regimes. Increased content of polyphenols was observed in the rain-fed fruit; particularly, anthocyanin concentration was higher at ripening. Several secondary metabolites were differentially accumulated between different irrigation conditions. These results showed that these metabolic approaches could be efficiently used to determine the effects of agronomic treatments on olive fruit physiology and, consequently, on nutritional and healthy properties of the obtained extra-virgin olive oil.

Keywords: olea europea, anthocyanins, polyphenols, water regimes

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Authors: M. R. Haji Hajikolaei, A. A. Nikvand, A. R. Ghadrdan, M. Ghorbanpoor, B. Mohammadian

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This study was carried out on water buffalo for detection of Leptospira interrogans in kidney and urine and its relationship with serological findings. Blood, urine and kidney samples were taken immediately after slaughter from 353 water buffalos at Ahvaz abattoir in Khouzestan province, Iran. Sera were initially screened at serum dilution of 1:100 against seven live antigens of Leptospira interrogans: pomona, hardjo, ballum, icterohemorrhagiae, tarasovi, australis and grippotyphosa using the microscopic agglutination test (MAT) and sera with positive results were titrated against reacting antigens in serial twofold dilution from 1:100 to 1:800. The samples of kidney were embedded in paraffin wax and 5µm thick sections were stained routinely with Haematoxylin and Eosin (H&E). Polymerase chain reaction (PCR) examination was done on urine and kidney by using LipL32 gene primers. Antibodies against one or more serovars at dilution >:100 were detected in sera. The most frequent reactor was hardjo (56.2%), followed by pomona (52.3%), australis (9.8%), tarassovi (5.9%), grippotyphosa (4.5%) and icterohaemorrhagiae (3.9%). The L. interrogans were detected in 43 (12.2%) of examined buffaloes, so that 26 (8.2%) of kidney tissues, 14 (4.8%) of urine samples separately and 3 (0.84%) of both kidney and urine samples were positive in PCR. From 153 (43.3%) buffaloes with positive MAT, 24 cases were positive by PCR of kidney and/or urine samples, synchronously. Renal lesions such as interstitial nephritis, acute tubular necrosis (ATN), pyelonephritis, glomerolonephritis, renal fibrosis and hydronephrosis were found in 128 (36.3%) cases. Statistical analysis indicated that there was no significant association between results of MAT, PCR and interstitial nephritis.

Keywords: leptospiral infection, PCR, MAT, histopathology, river buffalo

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Authors: Suwattana Visetnan, Premruethai Supungul, Sureerat Tang, Ikuo Hirono, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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In animals, infection by Gram-negative bacteria and certain viruses activates the Imd signaling pathway wherein the a NF-κB transcription factor, Relish, is a key regulatory protein for the synthesis of antimicrobial proteins. Infection by yellow head virus (YHV) activates the Imd pathway. To investigate the expression of genes involved in YHV infection and under the influence of PmRelish regulation, RNA interference and suppression subtractive hybridization (SSH) are employed. The genes in forward library expressed in shrimp after YHV infection and under the activity of PmRelish were obtained by subtracting the cDNAs from YHV-infected and PmRelish-knockdown shrimp with cDNAs from YHV-infected shrimp. Opposite subtraction gave a reverse library whereby an alternative set of genes under YHV infection and no PmRelish expression was obtained. Sequencing of 252 and 99 cDNA clones from the respective forward and reverse libraries were done and annotated through blast search against the GenBank sequences. Genes involved in defense and homeostasis were abundant in both libraries, 31% and 23% in the forward and reverse libraries, respectively. They were predominantly antimicrobial proteins, proteinases and proteinase inhibitors. The expression of antimicrobial protein genes, ALFPm3, crustinPm1, penaeidin3 and penaeidin5 were tested under PmRelish silencing and Gram-negative bacterium V. harveyi infection. Together with the results previously reported, the expression of penaeidin5 and also penaeidin3 but not ALFPm3 and crustinPm1 were under the regulation of PmRelish in the Imd pathway.

Keywords: relish, yellow head virus, penaeus monodon, antimicrobial proteins

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Authors: Saskya E. Carrera P., Ben Hankamer, Melanie Oey

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The green alga C. reinhardtii provides a platform for the cheap, scalable, and safe production of complex proteins. Despite gene expression in photosynthetic organisms being tightly regulated by light, most expression studies have analysed chloroplast recombinant protein production under constant light. Here the influence of illumination time and intensity on GFP and a GFP-PlyGBS (bacterial-lysin) fusion protein expression was investigated. The expression of both proteins was strongly influenced by the light regime (6-24 hr illumination per day), the light intensity (0-450 E m⁻²s⁻¹) and growth condition (photoautotrophic, mixotrophic and heterotrophic). Heterotrophic conditions resulted in relatively low recombinant protein yields per unit volume, despite high protein yields per cell, due to low growth rates. Mixotrophic conditions exhibited the highest yields at 6 hrs illumination at 200µE m⁻²s⁻¹ and under continuous low light illumination (13-16 mg L⁻¹ GFP and 1.2-1.6 mg L⁻¹ GFP-PlyGBS), as these conditions supported good cell growth and cellular protein yields. A ~23-fold increase in protein accumulation per cell and ~9-fold increase L⁻¹ culture was observed compared to standard constant 24 hr illumination for GFP-PlyGBS. The highest yields under photoautotrophic conditions were obtained under 9 hrs illumination (6 mg L⁻¹ GFP and 2.1 mg L⁻¹ GFP-PlyGBS). This represents a ~4-fold increase in cellular protein accumulation for GFP-PlyGBS. On a volumetric basis the highest yield was at 15 hrs illumination (~2-fold increase L⁻¹ over the constant light for GFP-PlyGBS). Optimising illumination conditions to balance growth and protein expression can thus significantly enhance overall recombinant protein production in C. reinhardtii cultures.

Keywords: chlamydomonas reinhardtii, light, mixotrophic, recombinant protein

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Authors: Nesrine Lenchi, Salima Kebbouche-Gana, Laddada Belaid, Mohamed Lamine Khelfaoui, Mohamed Lamine Gana

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Saline lakes are extreme hypersaline environments that are considered five to ten times saltier than seawater (150 – 300 g L-1 salt concentration). Hypersaline regions differ from each other in terms of salt concentration, chemical composition and geographical location, which determine the nature of inhabitant microorganisms. In order to explore the diversity of moderate and extreme halophiles Bacteria in Chott Tinsilt (East of Algeria), an isolation program was performed. In the first time, water samples were collected from the saltern during pre-salt harvesting phase. Salinity, pH and temperature of the sampling site were determined in situ. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions. Isolates were obtained by plating out the samples in complex and synthetic media. In this study, seven halophiles cultures of Bacteria were isolated. Isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (oxidase, catalase, nitrate reductase and urease), and optimization of growth conditions were done. The results indicated that the salinity optima varied from 50 to 250 g L-1, whereas the optimum of temperature range from 25°C to 35°C. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. The results showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Idiomarina, Halobacillus Thalassobacillus and Planococcus genera and what may represent a new bacterial genus.

Keywords: bacteria, Chott, halophilic, 16S rRNA

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Authors: Rameshwar Singh Seema

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In today’s world where diabetes has become an epidemic, our aim was to potentiate the effect of probiotics by integrating probiotics with cereals to formulate composite foods using Lactobacillus rhamnosus GG (LGG) and Lactobacillus casei NCDC19 against type 2 diabetes. After optimizing the product by Response Surface Methodology, it was studied for their effect on induction and progression of type 2 diabetes in HFD-fed Wistar rats. After 9 weeks study, best results were shown by the group fed with oat and milk based product fermented with LGG and L. casei NCDC19 which resulted in a significant decrease in blood glucose, HBA1c, improved OGTT, oxidative stress, cholesterol and triglycerides level during progression study of type 2 diabetes. During induction study also, there was significant reduction in blood glucose level, oxidative stress, cholesterol level and triglycerides level but slightly less as compared to progression study. Real time PCR gene expression studies were done for 5 genes (GLUT-4, IRS-2, ppar-γ, TNF-α, IL-6) whose expression is directly related to type 2 diabetes. The relative fold change expression was increased in case of GLUT-4, IRS-2, ppar-γ and decreased in case of TNF-α and IL-6 during both induction and progression study of diabetes but more significantly during progression study. Hence it was concluded that oat and milk based probiotic fermented product showed the synergistic effect of probiotics and oats especially in case of progression of type 2 diabetes. The benefits of these probiotic formulations may be further validated by clinical trials.

Keywords: type 2 diabetes, LGG, L.casei NCDC19, food science

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Authors: Ghulam Muhammad Ali

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Unveiling of genes involved in drought and root architecture using transcriptomic analyses remained fragmented for further improvement of wheat through genome editing. The purpose of this research endeavor was to unveil the variations in different genes implicated in drought tolerance and root architecture in wheat through RNA-seq data analysis. In this study seedlings of 8 days old, 6 cultivars of wheat namely, Batis, Blue Silver, Local White, UZ888, Chakwal 50 and Synthetic wheat S22 were subjected to transcriptomic analysis for root and shoot genes. Total of 12 RNA samples was sequenced by Illumina. Using updated wheat transcripts from Ensembl and IWGC references with 54,175 gene models, we found that 49,621 out of 54,175 (91.5%) genes are expressed at an RPKM of 0.1 or more (in at least 1 sample). The number of genes expressed was higher in Local White than Batis. Differentially expressed genes (DEG) were higher in Chakwal 50. Expression-based clustering indicated conserved function of DRO1and RPK1 between Arabidopsis and wheat. Dendrogram showed that Local White is sister to Chakwal 50 while Batis is closely related to Blue Silver. This study flaunts transcriptomic sequence variations in different cultivars that showed mutations in genes associated with drought that may directly contribute to drought tolerance. DRO1 and RPK1 genes were fetched/isolated for genome editing. These genes are being edited in wheat through CRISPR-Cas9 for yield enhancement.

Keywords: transcriptomic, wheat, genome editing, drought, CRISPR-Cas9, yield enhancement

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Authors: S. Bahrami, A. Rezaie, Z. Boroumand, S. Ghavami

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Neospora caninum is protozoan parasite which can cause a serious disease in dogs and cattle. It has been shown that birds may be a permissive intermediate host for N. caninum since parasite DNA has been detected in tissues from birds. It is showed that embryonated chicken egg can be used as an animal model for experimental infection. The aim of present study was to compare experimental infection of Neospora in chicken and pigeons embryonated eggs. An infection with N. caninum Nc1 isolate was conducted in chicken and pigeons embryonated eggs to evaluate LD50. After calculation of LD50, 2LD50 of tachyzoites were injected to eggs. Macroscopic changes of each embryo were noticed and to investigate the parasite distribution in tissues immunohistochemistry (IHC) and molecular methods were used. In the present study, histopathological changes were considered and sections to those used for histopathological examination including heart, liver, brain and chorioallantoic (CA) membrane were subjected to IHC, too. For PCR procedure, primer pair Np21/Np6 was used for amplification of the Nc5 gene. Pigeon's embryo showed more macroscopic changes than chicken embryo. A hemorrhage of the CA was the main grass lesion. All the infected tissues had histopathological changes. Microscopic examination of tissues revealed acute neosporosis due to hemorrhage, necrosis and infiltration of mononuclear inflammatory cells. Based on IHC and molecular results, the parasite aggregation in the heart was more predominant than in the other tissues. These results reinforce that there is genetic susceptibility to N. caninum in pigeons embryonated eggs like chickens embryonated eggs and provide new insights to research an inexpensive and available animal model for N. caninum.

Keywords: immunohistochemistry, Neospora caninum, PCR, pigeon embryonated egg

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Authors: Maryam Torabi, Habibi, Abdolahi, Mohammadi, Hassanzadeh, Darban Maghami, Baghi

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Sheeppox Virus (SPPV) and goatpox virus (GTPV) are considerable diseases of sheep, and goats, caused by viruses of the Capripoxvirus (CaPV) genus. They are responsible for economic losses. Animal mortality, morbidity, cost of vaccinations, and restrictions in animal products’ trade are the reasons of economic losses. Control and eradication of CaPV depend on early detection of outbreaks so that molecular detection and genetic analysis could be effective to this aim. This study was undertaken to molecularly characterize SPPV and GTPV strains that have been circulating in Iran. 120 skin papules and nodule biopsies were collected from different regions of Iran and were examined for SPPV, GTPV viruses using TaqMan Real -Time PCR. Some of these amplified genes were sequenced, and phylogenetic trees were constructed. Out of the 120 samples analysed, 98 were positive for CaPV by Real- Time PCR (81.6%), and most of them wereSPPV. then 10 positive samples were sequenced and characterized by amplifying the ORF 103CaPV gene. sequencing and phylogenetic analysis for these positive samples revealed a high percentage of identity with SPPV isolated from different countries in Middle East. In conclusions, molecular characterization revealed nearly complete identity with all recent SPPVs strains in local countries that requires further studies to monitor the virus evolution and transmission pathways to better understand the virus pathobiology that will help for SPPV control.

Keywords: molecular epidemiology, Real-Time PCR, phylogenetic analysis, capripoxviruses

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Authors: Anahit Hakobjanyan, Zdenka Navratilova, Gabriela Strakova, Martin Petrek

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Aging has a suppressive influence on human immune cells. Apoptosis may play important role in age-dependent immunosuppression and lymphopenia. Prevention of apoptosis may be promoted by BCL2-dependent and BCL2-independent manner. BCL2 is an antiapoptotic factor that has an antioxidative role by locating the glutathione at mitochondria and repressing oxidative stress. STAT3 may suppress apoptosis in BCL2-independent manner and promote cell survival blocking cytochrome-c release and reducing ROS production. The aim of our study was to estimate the influence of aging on BCL2-dependent and BCL2-independent prevention of apoptosis via measurement of BCL2 and STAT3 mRNAs expressions. The study was done on Armenian population (2 groups: 37 healthy young (mean age±SE; min/max age, male/female: 37.6±1.1; 20/54, 15/22), 28 healthy aged (66.7±1.5; 57/85, 12/16)). mRNA expression in peripheral blood leukocytes (PBL) was determined by RT-PCR using PSMB2 as the reference gene. Statistical analysis was done with Graph-Pad Prism 5; P < 0.05 considered as significant. The expression of BCL2 mRNA was lower in aged group (0.199) compared with young ones (0.643)(p < 0.01). Decrease expression was also recorded for female and male subgroups (p < 0.01). The expression level of STAT3 mRNA was increased (young, 0.228; aged, 0.428) (p < 0.05) during aging (in the whole age group and male/female subgroups). Decreased level of BCL2 mRNA may indicate about the suppression of BCL2-dependent prevention of apoptosis during aging in peripheral blood leukocytes. At the same time increased the level of STAT3 may suggest about activation of BCL2-independent prevention of apoptosis during aging.

Keywords: BCL2, STAT3, aging, apoptosis

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Authors: Wei Wang, Yuan Hu

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Epoxy resins (EP), one of the most important thermosetting polymers, is widely applied in various fields due to its desirable properties, such as excellent electrical insulation, low shrinkage, outstanding mechanical stiffness, satisfactory adhesion and solvent resistance. However, like most of the polymeric materials, EP has the fatal drawbacks including inherent flammability and high yield of toxic smoke, which restricts its application in the fields requiring fire safety. So, it is still a challenge and an interesting subject to develop new flame retardants which can not only remarkably improve the flame retardancy, but also render modified resins low toxic gases generation. In recent work, polymer nanocomposites based on nanohybrids that contain two or more kinds of nanofillers have drawn intensive interest, which can realize performance enhancements. The realization of previous hybrids of carbon nanotubes (CNTs) and molybdenum disulfide provides us a novel route to decorate layered double hydroxide (LDH) nanosheets on the surface of β-FeOOH nanorods; the deposited LDH nanosheets can fill the network and promote the work efficiency of β-FeOOH nanorods. Moreover, the synergistic effects between LDH and β-FeOOH can be anticipated to have potential applications in reducing fire hazards of EP composites for the combination of condense-phase and gas-phase mechanism. As reported, β-FeOOH nanorods can act as a core to prepare hybrid nanostructures combining with other nanoparticles through electrostatic attraction through layer-by-layer assembly technique. In this work, LDH nanosheets wrapped β-FeOOH nanorods (LDH-β-FeOOH) hybrids was synthesized by a facile method, with the purpose of combining the characteristics of one dimension (1D) and two dimension (2D), to improve the fire resistance of epoxy resin. The hybrids showed a well dispersion in EP matrix and had no obvious aggregation. Thermogravimetric analysis and cone calorimeter tests confirmed that LDH-β-FeOOH hybrids into EP matrix with a loading of 3% could obviously improve the fire safety of EP composites. The plausible flame retardancy mechanism was explored by thermogravimetric infrared (TG-IR) and X-ray photoelectron spectroscopy. The reasons were concluded: condense-phase and gas-phase. Nanofillers were transferred to the surface of matrix during combustion, which could not only shield EP matrix from external radiation and heat feedback from the fire zone, but also efficiently retard transport of oxygen and flammable pyrolysis.

Keywords: fire hazards, toxic gases, self-assembly, epoxy

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Authors: Ghaleb Bin Huraib

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Atopic dermatitis (AD), also known as atopic eczema, is a chronic inflammatory skin disease characterized by severe itching and recurrent, relapsing eczema-like skin lesions, affecting up to 20% of children and 10% of adults in industrialized countries. AD is a complex multifactorial disease, and its exact etiology and pathogenesis have not been fully elucidated. The aim of this study was to investigate the impact of gene polymorphisms of T helper cell subtype Th1 and Th2 cytokines, interferon-gamma (IFN-γ), interleukin-6 (IL-6) and transforming growth factor (TGF)-β1on AD susceptibility in a Saudi cohort. One hundred four unrelated patients with AD and 195 healthy controls were genotyped for IFN-γ (874A/T), IL-6 (174G/C) and TGF-β1 (509C/T) polymorphisms using ARMS-PCR and PCR-RFLP technique. The frequency of genotypes AA and AT of IFN-γ (874A/T) differed significantly among patients and controls (P 0.001). The genotype AT was increased while genotype AA was decreased in AD patients as compared to controls. AD patients also had a higher frequency of T-containing genotypes (AT+TT) than controls (P = 0.001). The frequencies of alleles T and A were statistically different in patients and controls (P = 0.04). The frequencies of genotype GG and allele G of IL-6 (174G/C) were significantly higher, while genotype GC and allele C were lower in AD patients than in controls. There was no significant difference in the frequencies of alleles and genotypes of TGF-β1 (509C/T) polymorphism between the patient and control groups. These results showed that susceptibility to AD is influenced by the presence or absence of genotypes of IFN-γ (874A/T) and IL-6 (174G/C) polymorphisms. It is concluded T-allele and T-containing genotypes (AT+TT) of IFN-γ (874A/T) and G-allele and GG genotype ofIL-6 (174G/C) polymorphisms are susceptible to AD in Saudis. On the other hand, the TGF-β1 (509C/T) polymorphism may not be associated with AD risk in our population; however, further studies with large sample sizes are required to confirm these results.

Keywords: atopic dermatitis, Polymorphism, Interferon, IL-6

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Authors: Mohammed N. Al Kindi, Mazin Al Khabouri, Khalsa Al Lamki, Tommasso Pappuci, Giovani Romeo, Nadia Al Wardy

Abstract:

Hereditary hearing loss is a heterogeneous group of complex disorders with an overall incidence of one in every five hundred newborns presented as syndromic and non-syndromic forms. Cadherin-related 23 (CDH23) is one of the listed deafness causative genes. CDH23 is found to be expressed in the stereocilia of hair cells and the retina photoreceptor cells. Defective CDH23 has been associated mostly with prelingual severe-to-profound sensorineural hearing loss (SNHL) in either syndromic (USH1D) or non-syndromic SNHL (DFNB12). An Omani family diagnosed clinically with severe-profound sensorineural hearing loss was genetically analysed by whole exome sequencing technique. A novel homozygous missense variant, c.A7451C (p.D2484A), in exon 53 of CDH23 was detected. One hundred and thirty control samples were analysed where all were negative for the detected variant. The variant was analysed in silico for pathogenicity verification using several mutation prediction software. The variant proved to be a pathogenic mutation and is reported for the first time in Oman and worldwide. It is concluded that in silico mutation prediction analysis might be used as a useful molecular diagnostics tool benefiting both genetic counseling and mutation verification. The aspartic acid 2484 alanine missense substitution might be the main disease-causing mutation that damages CDH23 function and could be used as a genetic hearing loss marker for this particular Omani family.

Keywords: Cdh23, d2484a, in silico, Oman

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Authors: Nafiseh Noormohammadi, Ahmad Sobhani Najafabadi

Abstract:

Hypericins (hypericin and pseudohypericin) are natural napthodianthrone derivatives produced by Hypericum perforatum (St. John’s Wort), which have many medicinal properties such as antitumor, antineoplastic, antiviral, and antidepressant activities. Production and accumulation of hypericin in the plant are influenced by both genetic and environmental conditions. Despite the existence of different high-throughput data on the plant, genetic dimensions of hypericin biosynthesis have not yet been completely understood. In this research, 21 high-quality RNA-seq data on different parts of the plant were integrated into metabolic data to reconstruct a coexpression network. Results showed that a cluster of 30 transcripts was correlated with total hypericin. The identified transcripts were divided into three main groups based on their functions, including hypericin biosynthesis genes, transporters, detoxification genes, and transcription factors (TFs). In the biosynthetic group, different isoforms of polyketide synthase (PKSs) and phenolic oxidative coupling proteins (POCPs) were identified. Phylogenetic analysis of protein sequences integrated into gene expression analysis showed that some of the POCPs seem to be very important in the biosynthetic pathway of hypericin. In the TFs group, six TFs were correlated with total hypericin. qPCR analysis of these six TFs confirmed that three of them were highly correlated. The identified genes in this research are a rich resource for further studies on the molecular breeding of H. perforatum in order to obtain varieties with high hypericin production.

Keywords: hypericin, St. John’s Wort, data mining, transcription factors, secondary metabolites

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Authors: Rima Zakzouk, Yasushi Shimada, Yasunori Sumi, Junji Tagami

Abstract:

Background and purpose: The resin composite has become the main material for the restorations of caries in recent years due to aesthetic characteristics, especially with the development of the adhesive techniques. The quality of adhesion to tooth structures is depending on an exchange process between inorganic tooth material and synthetic resin and a micromechanical retention promoted by resin infiltration in partially demineralized dentin. Optical coherence tomography (OCT) is a noninvasive diagnostic method for obtaining cross-sectional images that produce high-resolution of the biological tissue at the micron scale. The aim of this study was to evaluate the gap formation at adhesive/tooth interface of two-step self-etch adhesives that are preceded with or without phosphoric acid pre-etching in different regions of teeth using SS-OCT. Materials and methods: Round tapered cavities (2×2 mm) were prepared in cervical part of bovine incisors teeth and divided into 2 groups (n=10): first group self-etch adhesive (Clearfil SE Bond) was applied for SE group and second group treated with acid etching before applying the self-etch adhesive for PA group. Subsequently, both groups were restored with Estelite Flow Quick Flowable Composite Resin and observed under OCT. Following 5000 thermal cycles, the same section was obtained again for each cavity using OCT at 1310-nm wavelength. Scanning was repeated after two months to monitor the gap progress. Then the gap length was measured using image analysis software, and the statistics analysis were done between both groups using SPSS software. After that, the cavities were sectioned and observed under Confocal Laser Scanning Microscope (CLSM) to confirm the result of OCT. Results: Gaps formed at the bottom of the cavity was longer than the gap formed at the margin and dento-enamel junction in both groups. On the other hand, pre-etching treatment led to damage the DEJ regions creating longer gap. After 2 months the results showed almost progress in the gap length significantly at the bottom regions in both groups. In conclusions, phosphoric acid etching treatment did not reduce the gap lrngth in most regions of the cavity. Significance: The bottom region of tooth was more exposed to gap formation than margin and DEJ regions, The DEJ damaged with phosphoric acid treatment.

Keywords: optical coherence tomography, self-etch adhesives, bottom, dento enamel junction

Procedia PDF Downloads 227

Authors: Aliaa M. El-Borai, Ehab A. Beltagy, Eman E. Gadallah, Samy A. ElAssar

Abstract:

Bioremediation technology is now used for treatment instead of traditional metal removal methods. A strain was isolated from Marsa Alam, Red sea, Egypt showed high resistance to high lead concentration and was identified by the 16S rRNA gene sequencing technique as Halomonas sp. ES015. Medium optimization was carried out using Plackett-Burman design, and the most significant factors were yeast extract, casamino acid and inoculums size. The optimized media obtained by the statistical design raised the removal efficiency from 84% to 99% from initial concentration 250 ppm of lead. Moreover, Box-Behnken experimental design was applied to study the relationship between yeast extract concentration, casamino acid concentration and inoculums size. The optimized medium increased removal efficiency to 97% from initial concentration 500 ppm of lead. Immobilized Halomonas sp. ES015 cells on sponge cubes, using optimized medium in loop bioremediation column, showed relatively constant lead removal efficiency when reused six successive cycles over the range of time interval. Also metal removal efficiency was not affected by flow rate changes. Finally, the results of this research refer to the possibility of lead bioremediation by free or immobilized cells of Halomonas sp. ES015. Also, bioremediation can be done in batch cultures and semicontinuous cultures using column technology.

Keywords: bioremediation, lead, Box–Behnken, Halomonas sp. ES015, loop bioremediation, Plackett-Burman

Procedia PDF Downloads 196

Authors: Nazanin Amanat, Alison Tayler, Steve Whyard

Abstract:

While chemical insecticides effectively control many insect pests, they also harm many non-target species. Double-stranded RNA (dsRNA) pesticides, in contrast, can be designed to target unique gene sequences and thus act in a species-specific manner. DsRNA insecticides do not, however, work equally well for all insects, and for some species that are considered refractory to dsRNA, a primary factor affecting efficacy is the relative ease by which dsRNA can enter a target cell’s cytoplasm. In this study, we are examining how different structured dsRNAs (linear, hairpin, and paperclip) can enter mosquito and lepidopteran cells, as they represent dsRNA-sensitive and refractory species, respectively. To determine how the dsRNAs enter the cells, we are using chemical inhibitors and RNA interference (RNAi)-mediated knockdown of key proteins associated with different endocytosis processes. Understanding how different dsRNAs enter cells will ultimately help in the design of molecules that overcome refractoriness to RNAi or develop resistance to dsRNA-based insecticides. To date, we have conducted chemical inhibitor experiments on both cell lines and have evidence that linear dsRNAs enter the cells using clathrin-mediated endocytosis, while the paperclip dsRNAs (pcRNAs) can enter both species’ cells in a clathrin-independent manner to induce RNAi. An alternative uptake mechanism for the pcRNAs has been tentatively identified, and the outcomes of our RNAi-mediated knockdown experiments, which should provide corroborative evidence of our initial findings, will be discussed.

Keywords: dsRNA, RNAi, uptake, insecticides, dipteran, lepidopteran

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Authors: M. Latha, Achintya K. Dolui, P. Vijayaraj

Abstract:

Vegetable oils mainly triacylglycerol (TAG) are an essential nutrient in the human diet as well as one of the major global commodity. There is a pressing need to enhance the yield of oil production to meet the world’s growing demand. Oil content is controlled by the balance between synthesis and breakdown in the cells. Several studies have established to increase the oil content by the overexpression of oil biosynthetic enzymes. Interestingly the significant oil accumulation was observed with impaired TAG hydrolysis. Unfortunately, the structural, as well as the biochemical properties of the lipase enzymes, is widely unknown, and so far, no candidate gene was identified in seeds except sugar-dependent1 (SDP1). Evidence has shown that SDP1directly responsible for initiation of oil breakdown in the seeds during germination. The present study is the identification of seed-specific acyl-hydrolases by activity based proteome profiling (ABPP) using Arabidopsis thaliana as a model system. The ABPP reveals that around 8 to 10 proteins having the serine hydrolase domain and are expressed during germination of Arabidopsis seed. The N-term sequencing, as well as LC-MS/MS analysis, was performed for the differentially expressed protein during germination. The coding region of the identified proteins was cloned, and lipases activity was assessed with purified recombinant protein. The enzyme assay was performed against various lipid substrates, and we have observed the acylhydrolase activity towards lysophosphatidylcholine and monoacylglycerol. Further, the functional characteristic of the identified protein will reveal the physiological significance the enzyme in oil accumulation.

Keywords: lipase, lipids, vegetable oil, triacylglycerol

Procedia PDF Downloads 187

Authors: Zhang Lei, Zhao Qingrong, Wang Chen, Zhang Sufang, Zhang Hanguo

Abstract:

Larch is the main afforestation and timber tree species in Northeast China, and drought is one of the main factors limiting the growth of Larch and other organisms in Northeast China. In order to further explore the mechanism of Larch drought resistance, PEG was used to simulate drought stress. The full-length sequencing of Larch embryogenic callus under PEG simulated drought stress was carried out by combining Illumina-Hiseq and SMRT-seq. A total of 20.3Gb clean reads and 786492 CCS reads were obtained from the second and third generation sequencing. The de-redundant transcript sequences were predicted by lncRNA, 2083 lncRNAs were obtained, and the target genes were predicted, and a total of 2712 target genes were obtained. The de-redundant transcripts were further screened, and 1654 differentially expressed genes (DEGs )were obtained. Among them, different DEGs respond to drought stress in different ways, such as oxidation-reduction process, starch and sucrose metabolism, plant hormone pathway, carbon metabolism, lignin catabolic/biosynthetic process and so on. This study provides basic full-length sequencing data for the study of Larch drought resistance, and excavates a large number of DEGs in response to drought stress, which helps us to further understand the function of Larch drought resistance genes and provides a reference for in-depth analysis of the molecular mechanism of Larch drought resistance.

Keywords: larch, drought stress, full-length transcriptome sequencing, differentially expressed genes

Procedia PDF Downloads 172

Authors: Huajuan Shi

Abstract:

Background: The biopsy of the preimplantation embryo may increase the potential risk and concern of embryo viability. Clinically discarded spent embryo medium (SEM) has entered the view of researchers, sparking an interest in noninvasive embryo screening. However, one of the major restrictions is the extremelty low quantity of cf-RNA, which is difficult to efficiently and unbiased amplify cf-RNA using traditional methods. Hence, there is urgently need to an efficient and low bias amplification method which can comprehensively and accurately obtain cf-RNA information to truly reveal the state of SEM cf-RNA. Result: In this present study, we established an agarose PCR amplification system, and has significantly improved the amplification sensitivity and efficiency by ~90 fold and 9.29 %, respectively. We applied agarose to sequencing library preparation (named AG-seq) to quantify and characterize cf-RNA in SEM. The number of detected cf-RNAs (3533 vs 598) and coverage of 3' end were significantly increased, and the noise of low abundance gene detection was reduced. The increasing percentage 5' end adenine and alternative splicing (AS) events of short fragments (< 400 bp) were discovered by AG-seq. Further, the profiles and characterizations of cf-RNA in spent cleavage medium (SCM) and spent blastocyst medium (SBM) indicated that 4‐mer end motifs of cf-RNA fragments could remarkably differentiate different embryo development stages. Significance: This study established an efficient and low-cost SEM amplification and library preparation method. Not only that, we successfully described the characterizations of SEM cf-RNA of preimplantation embryo by using AG-seq, including abundance features fragment lengths. AG-seq facilitates the study of cf-RNA as a noninvasive embryo screening biomarker and opens up potential clinical utilities of trace samples.

Keywords: cell-free RNA, agarose, spent embryo medium, RNA sequencing, non-invasive detection

Procedia PDF Downloads 92

Authors: Asma Lamin, Anna H. Kaksonen, Ivan Cole, Paul White, Xiao-Bo Chen

Abstract:

Microbiologically influenced corrosion (MIC) is a process in which microorganisms initiate, facilitate, or accelerate the electrochemical corrosion reactions of metallic components. Several reports documented that MIC accounts for about 20 to 40 % of the total cost of corrosion. Biofilm formation due to the presence of microorganisms on the surface of metal components is known to play a vital role in MIC, which can lead to severe consequences in various environmental and industrial settings. Quorum sensing (QS) system plays a major role in regulating biofilm formation and control the expression of some microbial enzymes. QS is a communication mechanism between microorganisms that involves the regulation of gene expression as a response to the microbial cell density within an environment. This process is employed by both Gram-positive and Gram-negative bacteria to regulate different physiological functions. QS involves production, detection, and responses to signalling chemicals, known as auto-inducers. QS controls specific processes important for the microbial community, such as biofilm formation, virulence factor expression, production of secondary metabolites and stress adaptation mechanisms. The use of QS inhibitors (QSIs) has been proposed as a possible solution to biofilm related challenges in many different applications. Although QSIs have demonstrated some strength in tackling biofouling, QSI-based strategies to control microbially influenced corrosion have not been thoroughly investigated. As such, our research aims to target the QS mechanisms as a strategy for mitigating MIC on metal surfaces in engineered systems.

Keywords: quorum sensing, quorum quenching, biofilm, biocorrosion

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Authors: Pejman Taghibeikzadehbadr

Abstract:

Muscle contraction stimulates a transient change of myogenic factors, partly related to the mode of contractions. Here, we assessed the response of Insulin-like growth factor 1Ea (IGF-1Ea), Insulin-like growth factor 1Eb (IGF-1Eb), Insulin-like growth factor 1Ec (IGF-1Ec), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α-1), Peroxisome proliferator-activated receptor gamma coactivator 4-alpha (PGC1α-4), and myostatin to the eccentric Vs the concentric contraction in human skeletal muscle. Ten healthy males were performed an acute eccentric and concentric exercise bout (n = 5 per group). For each contraction type, participants performed 12 sets of 10 repetitions knee extension by the dominant leg. Baseline and post-exercise muscle biopsy were taken 4 weeks before and immediately after experimental sessions from Vastus Lateralis muscle. Genes expression was measured by real-time PCR technique. There was a significant increase in PGC1α-1, PGC1α-4, IGF-1Ea and, IGF-1Eb mRNA after concentric contraction (p ≤ 0.05), while the PGC1α-4 and IGF-1Ec significantly increased after eccentric contraction (p ≤ 0.05). It is intriguing to highlight that; no significant differences between groups were evident for changes in any variables following exercise bouts (p ≥ 0.05). Our results found that concentric and eccentric contractions presented different responses in PGC1α-1, IGF-1Ea, IGF-1Eb, and IGF-1Ec mRNA. However, a similar significant increase in mRNA content was observed in PGC1α-4. Further, no apparent differences could be found between the response of genes to eccentric and concentric contraction.

Keywords: eccentric contraction, concentric contraction, gene expression, PGC-1 alpha, IGF-1 Myostatin

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Authors: Michio Kurosu

Abstract:

Alterations in glycosylation not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Identification of cell type-specific glycoconjugates (tumor markers) has led to the discovery of new assay systems for certain cancers via immunodetection reagents. N- and O-linked glycans are the most abundant forms of glycoproteins. Recent studies of cancer immunotherapy are based on the immunogenicity of truncated O-glycan chains (e.g., Tn, sTn, T, and sLea/x). The prevalence of N-linked glycan changes in the development of tumor cells is known; however, therapeutic antibodies against N-glycans have not yet been developed. This is due to the lack of specificity of N-linked glycans between normal/healthy and cancer cells. Abnormal branching of N-linked glycans has been observed, particularly in solid cancer cells. While the discovery of drug-like glycosyltransferase inhibitors that block the biosynthesis of specific branching has a very low likelihood of success, altered glycosylation levels can be exploited by suppressing N-glycan biosynthesis through the inhibition of dolichyl-phosphate N-acetylglucosaminephosphotransferase1 (DPAGT1) activity. Inhibition of DPAGT1 function leads to changes of O-glycosylation on proteins associated with mitochondria and zinc finger binding proteins (indirect effects). On the basis of dynamic crosstalk between DPAGT1 and Snail/Slung/ZEB1 (a family of transcription factors that promote the repression of the adhesion molecules), we have developed pharmacologically acceptable selective DPAGT1 inhibitors. Tunicamycin kills a wide range of cancer and healthy cells in a non-selective manner. In sharp contrast, our DPAGT1 inhibitors display strong cytostatic effects against 16 solid cancers, which require the overexpression of DPAGT1 in their progression but do not affect the cell viability of healthy cells. The identified DPAGT1 inhibitors possess impressive anti-metastatic ability in various solid cancer cell lines and induce their mitochondrial structural changes, resulting in apoptosis. A prototype DPAGT1 inhibitor, APPB has already been proven to shrink solid tumors (e.g., pancreatic cancers, triple-negative breast cancers) in vivo while suppressing metastases and has strong synergistic effects when combined with current cytotoxic drugs (e.g., paclitaxel). At this conference, our discovery of selective DPAGT1 inhibitors with drug-like properties and proof-of-pharmaceutical concept studies of a novel DPAGT1 inhibitor are presented.

Keywords: DPAGT1 inhibitors, anti-metastatic drugs, natural product based drug designs, cytostatic effects

Procedia PDF Downloads 76

Authors: Huajuan Shi

Abstract:

Background: The biopsy of the preimplantation embryo may increase the potential risk and concern of embryo viability. Clinically discarded spent embryo medium (SEM) has entered the view of researchers, sparking an interest in noninvasive embryo screening. However, one of the major restrictions is the extremelty low quantity of cf-RNA, which is difficult to efficiently and unbiased amplify cf-RNA using traditional methods. Hence, there is urgently need to an efficient and low bias amplification method which can comprehensively and accurately obtain cf-RNA information to truly reveal the state of SEM cf-RNA. Result: In this present study, we established an agarose PCR amplification system, and has significantly improved the amplification sensitivity and efficiency by ~90 fold and 9.29 %, respectively. We applied agarose to sequencing library preparation (named AG-seq) to quantify and characterize cf-RNA in SEM. The number of detected cf-RNAs (3533 vs 598) and coverage of 3' end were significantly increased, and the noise of low abundance gene detection was reduced. The increasing percentage 5' end adenine and alternative splicing (AS) events of short fragments (< 400 bp) were discovered by AG-seq. Further, the profiles and characterizations of cf-RNA in spent cleavage medium (SCM) and spent blastocyst medium (SBM) indicated that 4‐mer end motifs of cf-RNA fragments could remarkably differentiate different embryo development stages. Significance: This study established an efficient and low-cost SEM amplification and library preparation method. Not only that, we successfully described the characterizations of SEM cf-RNA of preimplantation embryo by using AG-seq, including abundance features fragment lengths. AG-seq facilitates the study of cf-RNA as a noninvasive embryo screening biomarker and opens up potential clinical utilities of trace samples.

Keywords: cell-free RNA, agarose, spent embryo medium, RNA sequencing, non-invasive detection

Procedia PDF Downloads 64