Search results for: mecA gene

Authors: Alaa Abdellatif, Gabrièle Breda

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Background. Advanced therapy medicinal products (ATMPs) are innovative therapies that mainly target orphan diseases and high unmet medical needs. ATMP includes gene therapy medicinal products (GTMP), somatic cell therapy medicinal products (CTMP), and tissue-engineered therapies (TEP). Since legislation opened the way in 2007, 25 ATMPs have been approved in the EU, which is about the same amount as the U.S. Food and Drug Administration. However, not all of the ATMPs that have been approved have successfully reached the market and retained their approval. Objectives. We aim to understand all the factors limiting the market access to very promising therapies in a systemic approach, to be able to overcome these problems, in the future, with scientific, regulatory and commercial innovations. Further to recent reviews that focus either on specific countries, products, or dimensions, we will address all the challenges faced by ATMP development today. Methodology. We used mixed methods and a multi-level approach for data collection. First, we performed an updated academic literature review on ATMP development and their scientific and market access challenges (papers published between 2018 and April 2023). Second, we analyzed industry feedback from cell and gene therapy webinars and white papers published by providers and pharmaceutical industries. Finally, we established a comparative analysis of the regulatory guidelines published by EMA and the FDA for ATMP approval. Results: The main challenges in bringing these therapies to market are the high development costs. Developing ATMPs is expensive due to the need for specialized manufacturing processes. Furthermore, the regulatory pathways for ATMPs are often complex and can vary between countries, making it challenging to obtain approval and ensure compliance with different regulations. As a result of the high costs associated with ATMPs, challenges in obtaining reimbursement from healthcare payers lead to limited patient access to these treatments. ATMPs are often developed for orphan diseases, which means that the patient population is limited for clinical trials which can make it challenging to demonstrate their safety and efficacy. In addition, the complex manufacturing processes required for ATMPs can make it challenging to scale up production to meet demand, which can limit their availability and increase costs. Finally, ATMPs face safety and efficacy challenges: dangerous adverse events of these therapies like toxicity related to the use of viral vectors or cell therapy, starting material and donor-related aspects. Conclusion. As a result of our mixed method analysis, we found that ATMPs face a number of challenges in their development, regulatory approval, and commercialization and that addressing these challenges requires collaboration between industry, regulators, healthcare providers, and patient groups. This first analysis will help us to address, for each challenge, proper and innovative solution(s) in order to increase the number of ATMPs approved and reach the patients

Keywords: advanced therapy medicinal products (ATMPs), product development, market access, innovation

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Authors: Berengere Vire, Nicolas Salvetat, Yoann Lannay, Guillaume Marcellin, Siem Van Der Laan, Franck Molina, Dinah Weissmann

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Predicting suicidal behaviors is one of the most complex challenges of daily psychiatric practices. Today, suicide risk prediction using biological tools is not validated and is only based on subjective clinical reports of the at-risk individual. Therefore, there is a great need to identify biomarkers that would allow early identification of individuals at risk of suicide. Alterations of adenosine-to-inosine (A-to-I) RNA editing of neurotransmitter receptors and other proteins have been shown to be involved in etiology of different psychiatric disorders and linked to suicidal behavior. RNA editing is a co- or post-transcriptional process leading to a site-specific alteration in RNA sequences. It plays an important role in the epi transcriptomic regulation of RNA metabolism. On postmortem human brain tissue (prefrontal cortex) of depressed suicide victims, Alcediag found specific alterations of RNA editing activity on the mRNA coding for the serotonin 2C receptor (5-HT2cR). Additionally, an increase in expression levels of ADARs, the RNA editing enzymes, and modifications of RNA editing profiles of prime targets, such as phosphodiesterase 8A (PDE8A) mRNA, have also been observed. Interestingly, the PDE8A gene is located on chromosome 15q25.3, a genomic region that has recurrently been associated with the early-onset major depressive disorder (MDD). In the current study, we examined whether modifications in RNA editing profile of prime targets allow identifying disease-relevant blood biomarkers and evaluating suicide risk in patients. To address this question, we performed a clinical study to identify an RNA editing signature in blood of depressed patients with and without the history of suicide attempts. Patient’s samples were drawn in PAXgene tubes and analyzed on Alcediag’s proprietary RNA editing platform using next generation sequencing technology. In addition, gene expression analysis by quantitative PCR was performed. We generated a multivariate algorithm comprising various selected biomarkers to detect patients with a high risk to attempt suicide. We evaluated the diagnostic performance using the relative proportion of PDE8A mRNA editing at different sites and/or isoforms as well as the expression of PDE8A and the ADARs. The significance of these biomarkers for suicidality was evaluated using the area under the receiver-operating characteristic curve (AUC). The generated algorithm comprising the biomarkers was found to have strong diagnostic performances with high specificity and sensitivity. In conclusion, we developed tools to measure disease-specific biomarkers in blood samples of patients for identifying individuals at the greatest risk for future suicide attempts. This technology not only fosters patient management but is also suitable to predict the risk of drug-induced psychiatric side effects such as iatrogenic increase of suicidal ideas/behaviors.

Keywords: blood biomarker, next-generation-sequencing, RNA editing, suicide

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Authors: Morello Sara, Pederiva Sabina, Bianchi Manila, Martucci Francesca, Marchis Daniela, Decastelli Lucia

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Vegetables represent an integral part of a healthy diet due to their valuable nutritional properties and the growth in consumer demand in recent years is particularly remarkable for a diet rich in vitamins and micronutrients. However, plant-based products are involved in several food outbreaks connected to various sources of contamination and quite often, bacteria responsible for side effects showed high resistance to antibiotics. The abuse of antibiotics can be one of the main mechanisms responsible for increasing antibiotic resistance (AR). Plants grown for food use can be contaminated directly by spraying antibiotics on crops or indirectly by treatments with antibiotics due to the use of manure, which may contain both antibiotics and genes of antibiotic resistance (ARG). Antibiotic residues could represent a potential way of human health risk due to exposure through the consumption of plant-based foods. The presence of antibiotic-resistant bacteria might pose a particular risk to consumers. The present work aims to investigate through a multidisciplinary approach the occurrence of ARG by means of a biomolecular approach (PCR) and the prevalence of antibiotic residues using a multi residues LC-MS/MS method, both in different plant-based products. During the period from July 2020 to October 2021, a total of 74 plant samples (33 lettuces and 41 tomatoes) were collected from 57 farms located throughout the Piedmont area, and18 out of 74 samples (11 lettuces and 7 tomatoes) were selected to LC-MS/MS analyses. DNA extracted (ExtractME, Blirt, Poland) from plants used on crops and isolated bacteria were analyzed with 6 sets of end-point multiplex PCR (Qiagen, Germany) to detect the presence of resistance genes of the main antibiotic families, such as tet genes (tetracyclines), bla (β-lactams) and mcr (colistin). Simultaneous detection of 43 molecules of antibiotics belonging to 10 different classes (tetracyclines, sulphonamides, quinolones, penicillins, amphenicols, macrolides, pleuromotilines, lincosamides, diaminopyrimidines) was performed using Exion LC system AB SCIEX coupled to a triple quadrupole mass spectrometer QTRAP 5500 from AB SCIEX. The PCR assays showed the presence of ARG in 57% (n=42): tetB (4.8%; n=2), tetA (9.5%; n=4), tetE (2.4%; n=1), tetL (12%; n=5), tetM (26%; n=11), blaSHV (21.5%; n=9), blaTEM (4.8%; n =2) and blaCTX-M (19%; n=8). In none of the analyzed samples was the mcr gene responsible for colistin resistance detected. Results obtained from LC-MS/MS analyses showed that none of the tested antibiotics appear to exceed the LOQ (100 ppb). Data obtained confirmed the presence of bacterial populations containing antibiotic resistance determinants such as tet gene (tetracycline) and bla genes (beta-lactams), widely used in human medicine, which can join the food chain and represent a risk for consumers, especially with raw products. The presence of traces of antibiotic residues in vegetables, in concentration below the LOQ of the LC-MS/MS method applied, cannot be excluded. In conclusion, traces of antibiotic residues could be a health risk to the consumer due to potential involvement in the spread of AR. PCR represents a useful and effective approach to characterize and monitor AR carried by bacteria from the entire food chain.

Keywords: plant-based products, ARG, PCR, antibiotic residues

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Authors: Rohita Gupta, G. S. Brah, R. Verma, C. S. Mukhopadhayay

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Although chicken strains show differences in susceptibility to a number of diseases, the underlying immunological basis is yet to be elucidated. In the present study, heterophils were subjected to LPS stimulation and total RNA extraction, further differential gene expression was studied in broiler, layer and indigenous Aseel strain by Real Time RT-PCR at different time periods before and after induction. The expression of the 14 AvBDs and chTLR 1, 2, 3, 4, 5, 7, 15 and 21 was detectable in heterophils. The expression level of most of the AvBDs significantly increased (P<0.05) 3 hours post in vitro lipopolysaccharide challenge. Higher expression level and stronger activation of most AvBDs, NFkB-1 and IRF-3 in heterophils was observed, with the stimulation of LPS in layer compared to broiler, and in Aseel compared to both layer and broiler. This investigation will allow more refined interpretation of immuno-genetic basis of the variable disease resistance/susceptibility in divergent stock of chicken including indigenous breed. Moreover this study will be helpful in formulation of strategy for isolation of antimicrobial peptides from heterophils.

Keywords: differential expression, heterophils, cytokines, defensin, TLR

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Authors: Inas Raafat, Azza El Bassiouny, Waldemar L. Olszewsky, Nagui E. Mikhail, Mona Nossier, Nora E. I. El-Bassiouni, Mona Zoheiry, Houda Abou Taleb, Noha Abd El-Aal, Ali Baioumy, Shimaa Attia

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Background: Orthotopic liver transplantation is an established treatment for patients with severe acute and end-stage chronic liver disease. The shortage of donor organs continues to be the rate-limiting factor for liver transplantation throughout the world. Hepatocyte transplantation is a promising treatment for several liver diseases and can, also, be used as a "bridge" to liver transplantation in cases of liver failure. Aim of the work: This study was designed to develop a highly efficient protocol for isolation and transplantation of hepatocytes in experimental Lewis rat model to provide satisfactory guidelines for future application on humans.Materials and Methods: Hepatocytes were isolated from the liver by double perfusion technique and bone marrow cells were isolated by centrifugation of shafts of tibia and femur of donor Lewis rats. Recipient rats were subjected to sub-lethal dose of irradiation 2 days before transplantation. In a laparotomy operation the spleen was injected by freshly isolated hepatocytes and bone marrow cells were injected intravenously. The animals were sacrificed 45 day latter and splenic sections were prepared and stained with H & E, PAS AFP and Prox1. Results: The data obtained from this study showed that the double perfusion technique is successful in separation of hepatocytes regarding cell number and viability. Also the method used for bone marrow cells separation gave excellent results regarding cell number and viability. Intrasplenic engraftment of hepatocytes and live tissue formation within the splenic tissue were found in 70% of cases. Hematoxylin and eosin stained splenic sections from 7 rats showed sheets and clusters of cells among the splenic tissues. Periodic Acid Schiff stained splenic sections from 7 rats showed clusters of hepatocytes with intensely stained pink cytoplasmic granules denoting the presence of glycogen. Splenic sections from 7 rats stained with anti-α-fetoprotein antibody showed brownish cytoplasmic staining of the hepatocytes denoting positive expression of AFP. Splenic sections from 7 rats stained with anti-Prox1 showed brownish nuclear staining of the hepatocytes denoting positive expression of Prox1 gene on these cells. Also, positive expression of Prox1 gene was detected on lymphocytes aggregations in the spleens. Conclusions: Isolation of liver cells by double perfusion technique using collagenase buffer is a reliable method that has a very satisfactory yield regarding cell number and viability. The intrasplenic route of transplantation of the freshly isolated liver cells in an immunocompromised model was found to give good results regarding cell engraftment and tissue formation. Further studies are needed to assess function of engrafted hepatocytes by measuring prothrombin time, serum albumin and bilirubin levels.

Keywords: Lewis rats, hepatocytes, BMCs, transplantation, AFP, Prox1

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Authors: Foad Alzoughool

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Conflicting studies on the association between the rs5219 polymorphism and type 2 diabetes, some studies have confirmed a strong relationship between this variant and type2 diabetes, on the other hand, many studies denied the existence of this association. This study aimed to provide evidence about whether the rs5219 polymorphism has or hasn't a role as a risk factor for diabetes and meta-analysis to investigate the role of the control age group in the association. Genotyping of the rs5219 polymorphism was performed in a cohort of 266 healthy elderly subjects with a mean age (60.2 ± 5.1) with no history of diabetes (HbA1c < 6%) using standard Sanger sequencing methods. Lys/Lys alleles were detected in 20 persons (7.5%), Lys/Glu alleles in 96 persons (36.1%), and Glu/Glu in 150 persons (56.4%). The genotype distribution was consistent with Hardy–Weinberg equilibrium (P =0.7). Meta-analysis notably indicates no association between rs5219 polymorphism and type 2 diabetes in all studies used the younger age of the control group compared to the patient's age. In conclusion, our study sheds light on the importance of age factor among the control group recruited in case-control studies.

Keywords: Type 2 diabetes, rs5219 polymorphism, E23K, KCNJ11 gene

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Authors: Eric Bang

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Interactions between receptors and ligands are crucial for many essential biological processes, including neurotransmission and metabolism. Ligand-receptor analyses that examine cell behavior and interactions often utilize cell type-specific RNA expressions from single-cell RNA sequencing (scRNA-seq) data. Using CellPhoneDB, a public repository consisting of ligands, receptors, and ligand-receptor interactions, the cell-cell interactions were explored in a specific scRNA-seq dataset from kidney tissue and portrayed the results with dot plots and heat maps. Depending on the type of cell, each ligand-receptor pair was aligned with the interacting cell type and calculated the positori probabilities of these associations, with corresponding P values reflecting average expression values between the triads and their significance. Using single-cell data (sample kidney cell references), genes in the dataset were cross-referenced with ones in the existing CellPhoneDB dataset. For example, a gene such as Pleiotrophin (PTN) present in the single-cell data also needed to be present in the CellPhoneDB dataset. Using the single-cell transcriptomics data via slide-seq and reference data, the CellPhoneDB program defines cell types and plots them in different formats, with the two main ones being dot plots and heat map plots. The dot plot displays derived measures of the cell to cell interaction scores and p values. For the dot plot, each row shows a ligand-receptor pair, and each column shows the two interacting cell types. CellPhoneDB defines interactions and interaction levels from the gene expression level, so since the p-value is on a -log10 scale, the larger dots represent more significant interactions. By performing an interaction analysis, a significant interaction was discovered for myeloid and T-cell ligand-receptor pairs, including those between Secreted Phosphoprotein 1 (SPP1) and Fibronectin 1 (FN1), which is consistent with previous findings. It was proposed that an effective protocol would involve a filtration step where cell types would be filtered out, depending on which ligand-receptor pair is activated in that part of the tissue, as well as the incorporation of the CellPhoneDB data in a streamlined workflow pipeline. The filtration step would be in the form of a Python script that expedites the manual process necessary for dataset filtration. Being in Python allows it to be integrated with the CellPhoneDB dataset for future workflow analysis. The manual process involves filtering cell types based on what ligand/receptor pair is activated in kidney cells. One limitation of this would be the fact that some pairings are activated in multiple cells at a time, so the manual manipulation of the data is reflected prior to analysis. Using the filtration script, accurate sorting is incorporated into the CellPhoneDB database rather than waiting until the output is produced and then subsequently applying spatial data. It was envisioned that this would reveal wherein the cell various ligands and receptors are interacting with different cell types, allowing for easier identification of which cells are being impacted and why, for the purpose of disease treatment. The hope is this new computational method utilizing spatially explicit ligand-receptor association data can be used to uncover previously unknown specific interactions within kidney tissue.

Keywords: bioinformatics, Ligands, kidney tissue, receptors, spatial transcriptome

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Authors: Baljeet Singh Saharan

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Bioremediation is the demand of the day. Tannery and textile effluents/waste waters have lots of pollution due to presence of hexavalent Chromium. Methodologies used in the present investigations include isolation, cultivation and purification of bacterial strain. Further characterization techniques and 16S rRNA sequencing were performed. Efficient bacterial strain capable of reducing hexavalent chromium was obtained. The strain can be used for bioremediation of industrial effluents containing hexavalent Cr. A gram negative, rod shaped and yellowish pigment producing bacterial strain from tannery effluent was isolated using nutrient agar. The 16S rRNA gene sequence similarity indicated that isolate SA13A is associated with genus Luteimonas (99%). This isolate has been found to reduce 100% of hexavalent chromium Cr (VI) (100 mg L-1) 100% in 16 h. Growth conditions were optimized for Cr (VI) reduction. Maximum reduction was observed at a temperature of 37 °C and pH 8.0. Additionally, Luteimonas aestuarii SA13A showed resistance against various heavy metals like Cr+6, Cr+3, Cu+2, Zn+2, Co+2, Ni+2 and Cd+2 . Hence, Luteimonas aestuarii SA13A could be used as potent Cr (VI) reducing strain as well as significant bioremediator in heavy metal contaminated sites.

Keywords: bioremediation, chromium, eco-friendly, heavy metals

Procedia PDF Downloads 465

Authors: H. Sakamoto, Y. Nakagawara, S. Oguri

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Drought stress is a critical environmental factor that adversely affects crop productivity and quality. Because of their immobile nature, plants have evolved mechanisms to sense and respond to drought stress. We identified a novel locus of Arabidopsis, designated DRA1 (drought responsive ankyrin 1), whose disruption leads to increased drought stress tolerance. DRA1 encodes a transmembrane protein with an ankyrin repeat motif that has been implicated in diverse cellular processes such as signal transduction. RT-PCR analysis revealed that there were at least two splicing variants of DRA1 transcripts in wild type plants. In response to drought stress, the levels of DRA1 transcripts retaining second and third introns were increased, whereas these introns were removed under unstressed conditions. These results suggest that DRA1 protein may negatively regulate plant drought tolerance and that the expression of DRA1 is regulated in response to drought stress by alternative splicing.

Keywords: alternative splicing, ankyrin repeat, Arabidopsis, drought tolerance

Procedia PDF Downloads 325

Authors: Chung-Young Lee, Se-Hee Ahn, Ilhwan Kim, Du-Min Go, Dae-Yong Kim, Jun-Gu Choi, Youn-Jeong Lee, Jae-Hong Kim, Hyuk-Joon Kwon

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The polymerase of avian influenza A virus (AIV) is a heterotrimer composed of PB2, PB1 and PA. PB2 plays a role in overcoming the host barrier; however, the genetic prerequisites for avian PB2 to acquire mammalian pathogenic mutations have not been well elucidated. Here, we demonstrated that key amino acid mutations (I66M, I109V and I133V, collectively referred to as MVV) of prototypic avian PB2 increase the replication efficiency of recombinant PR8 virus carrying the mutated PB2 in both avian and mammalian hosts. The MVV mutations caused no weight loss in mice, but they did allow replication in infected lungs, and the viruses acquired fatal mammalian pathogenic mutations such as Q591R/K, E627K, or D701N in the infected lungs. The MVV mutations are located at the interfaces of the trimer and are predicted to increase the strength of this structure. Thus, gaining MVV mutations might be the first step for AIV to acquire mammalian pathogenicity. These results provide new insights into the evolution of AIV in birds and mammals.

Keywords: avian influenza A virus, prototypic PB2, polymerase activity, mammalian pathogenicity, first-step mutations

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Authors: Sung-Hee Hwang, Michael Lee

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Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas mediated genome editing. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence specific double-stranded DNA (dsDNA) cleavage. Interestingly, ATG5 KO cells clearly showed a greater proliferation rate than WT NIH 3T3 cells, implying that autophagy induction is cytotoxic. Also, the clonogenic survival of ATG5 KO cells was greater than WT cells. The MTT assay revealed that the cytotoxic effect of PP2 was weaker on ATG5 knockout cells than that WT cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. Furthermore, Cyto-ID autophagy assay also revealed that PP2 failed to induce autophagy in ATG5 knockout cells. Together, our findings suggest that the resistance to PP2 in ATG5 knockout cells is associated with defective autophagy.

Keywords: ATG5 knockout, Autophagy, CRISPR/Cas9, PP2

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Authors: Ibrahim Ilker Ozyigit, Ertugrul Filiz, Seda Birbilener, Semsettin Kulac, Zeki Severoglu

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In this study, we have performed genetic diversity analysis of Tilia tomentosa genotypes by using randomly amplified polymorphic DNA (RAPD) primers. A total of 28 genotypes, including 25 members from the urban ecosystem and 3 genotypes from forest ecosystem as outgroup were used. 8 RAPD primers produced a total of 53 bands, of which 48 (90.6 %) were polymorphic. Percentage of polymorphic loci (P), observed number of alleles (Na), effective number of alleles (Ne), Nei's (1973) gene diversity (h), and Shannon's information index (I) were found as 94.29 %, 1.94, 1.60, 0.34, and 0.50, respectively. The unweighted pair-group method with arithmetic average (UPGMA) cluster analysis revealed that two major groups were observed. The genotypes of urban and forest ecosystems showed a high genetic similarity between 28% and 92% and these genotypes did not separate from each other in UPGMA tree. Also, urban and forest genotypes clustered together in principal component analysis (PCA).

Keywords: Tilia tomentosa, genetic diversity, urban ecosystem, RAPD, UPGMA

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Authors: A. T. Adetunji, F. B. Lewu, R. Mundembe

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Plants require nitrogen (N) to support desired production levels. Nitrogen is available to plants in the form of nitrate or ammonium, which are transported into the cell with the aid of various transport proteins. Ammonium transporters (AMTs) play a role in the uptake of ammonium, the form in which nitrogen is preferentially absorbed by plants. Solanum tuberosum AMT1 (StAMT1) was characterized using molecular biology and bioinformatics methods. Nucleotide database sequences were used to design AMT1-specific primers which were used to amplify the AMT1 internal regions. Nucleotide sequencing, alignment and phylogenetic analysis assigned StAMT1 to the AMT1 family. The deduced amino acid sequences showed that StAMT1 is 92%, 83% and 76% similar to Solanum lycopersicum LeAMT1.1, Lotus japonicus LjAMT1.1 and Solanum lycopersicum LeAMT1.2 respectively. StAMT1 fragments were shown to correspond to the 5th - 10th trans-membrane domains. Residue StAMT1 D15 is predicted to be essential for ammonium transport, while mutations of StAMT1 S76A may further enhance ammonium transport.

Keywords: ammonium transporter, bioinformatics, nitrogen, primers, Solanum tuberosum

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Authors: Abdelazeem Algammal, Reham Abouelmaatti, Xiaokun Li, Jisheng Ma, Eman Abdelnaby, Wael Elfeil

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Toll-like receptors (TLRs) are the best understood of the innate immune receptors that detect infections in vertebrates. However, the fish TLRs also exhibit very distinct features and a large diversity, which is likely derived from their diverse evolutionary history and the distinct environments that they occupy. Little is known about the fish immune system structure. Our work was aimed to identify and clone the Nile tilapiaTLR-3 as a model of freshwater fish species; we cloned the full-length cDNA sequence of Nile tilapia (Oreochromis niloticus) TLR-3 and according to our knowledge, it is the first report illustrating tilapia TLR-3. The complete cDNA sequence of Nile tilapia TLR-3 was 2736 pair base and it encodes a polypeptide of 912 amino acids. Analysis of the deduced amino acid sequence indicated that Nile tilapia TLR-3 has typical structural features and main components of proteins belonging to the TLR family. Our results illustrate a complete and functional Nile tilapia TLR-3 and it is considered an ortholog of the other vertebrate’s receptor.

Keywords: Nile tilapia, TLR-3, cloning, gene expression

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Authors: Farrah Putri Salmanida, Mei-Li Wu, Rika Wahyuningtyas, Wen-Bin Chung, Hso-Chi Chaung, Ko-Tung Chang

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Within the field of immunotherapy, oncolytic virotherapy (OVT) employs dual approaches that directly eliminate tumor cells while preserving healthy ones and indirectly reprogram the tumor microenvironment (TME) to elicit antitumor responses. Within the TME, tumor associated macrophages (TAMs) manifest characteristics akin to those of anti-inflammatory M2 macrophages, thus earning the designation of M2-like TAMs. In prior research, two antigens denoted as A1 (g6Ld10T) and A3 (ORF6L5), derived from a complete sequence of ORF5 with partial sequence of ORF6 in Porcine Reproductive and Respiratory Syndrome Virus Genotype 2 (PRRSV-2), demonstrated the capacity to repolarize M2-type porcine alveolar macrophages (PAMs) into M1 phenotypes. In this study, we sought for utilizing OVT strategies by introducing A1 or A3 on TAMs to endow them with the anti-tumor traits of M1 macrophages while retaining their capacity to target cancer cells. Upon exposing human THP-1-derived M2 macrophages to a cross-species test with 2 µg/ml of either A1 or A3 for 24 hours, real time PCR revealed that A3, but not A1, treated cells exhibited upregulated gene expressions of M1 markers (CCR7, IL-1ß, CCL2, Cox2, CD80). These cells reacted to virus-derived antigen, as evidenced by increased expression of pattern-recognition receptors TLR3, TLR7, and TLR9, subsequently providing feedback in the form of type I interferon responses like IFNAR1, IFN-ß, IRF3, IRF7, OAS1, Mx1, and ISG15. Through an MTT assay, only after 15 µg/ml of A3 treatment could the cell viability decrease, with a predicted IC50 of 16.96 µg/ml. Interestingly, A3 caused dose-dependent toxicity to a rat C6 glial cancer cell line even at doses as low as 2.5 µg/ml and reached its IC50 at 9.419 µg/ml. Using Annexin V/7AAD staining and PCR test, we deduced that a significant proportion of C6 cells were undergoing the early apoptosis phase predominantly through the intrinsic apoptosis cascade involving Bcl-2 family proteins. Following this stage, we conducted a test on A3’s repolarization ability, which revealed a significant rise in M1 gene expression markers, such as TNF, CD80, and IL-1ß, in M2-like TAMs generated in vitro from murine RAW264.7 macrophages grown with conditioned medium of 4T1 breast cancer cells. This was corroborated by the results of transcriptome analysis, which revealed that the primary subset among the top 10 to top 30 significantly upregulated differentially expressed genes (DEGs) dominantly consisted of M1 macrophages profiles, including Ccl3, Ccl4, Csf3, TNF, Bcl6b, Stc1, and Dusp2. Our findings unveiled the remarkable potential of the PRRSV-derived antigen A3 to repolarize macrophages while also being capable of selectively inducing apoptosis in cancerous cells. While further in vivo study is needed for A3, it holds promise as an adjuvant by its dual effects in cancer therapy modalities.

Keywords: cancer cell apoptosis, interferon responses, macrophage repolarization, recombinant protein

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Authors: Mariam Khaled, Noha Farag, Ghada Mohamed Abdel Salam, Khaled Abu-Aisha, Mohamed El-Azizi

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Gastric and colorectal cancers are among the most frequent causes of cancer-associated mortalities in Africa. They have been considered as a global public health concern, as nearly one million new cases are reported per year. IL-1β is a pro-inflammatory cytokine-produced by activated macrophages and monocytes- and a member of the IL-1 family. The inactive IL-1β precursor is cleaved and activated by caspase-1 enzyme, which itself is activated by the assembly of intracellular structures defined as NLRP3 (Nod Like receptor P3) inflammasomes. Activated IL-1β stimulates the Interleukin-1 receptor type-1 (IL-1R1), which is responsible for the initiation of a signal transduction pathway leading to cell proliferation. It has been proven that the IL-1β gene is a highly polymorphic gene in which single nucleotide polymorphisms (SNPs) may affect its expression. It has been previously reported that SNPs including base transitions between C and T at positions, -511 (C-T; dbSNP: rs16944) and -31 (C-T; dbSNP: rs1143627), from the transcriptional start site, contribute to the pathogenesis of gastric and colorectal cancers by affecting IL-1β levels. Altered production of IL-1β due to such polymorphisms is suspected to stimulate an amplified inflammatory response and promote Epithelial Mesenchymal Transition leading to malignancy. Allele frequency distribution of the IL-1β-31 and -511 SNPs, in different populations, and their correlation to the incidence of gastric and colorectal cancers, has been intriguing to researchers worldwide. The current study aims to investigate allele distributions of the IL-1β SNPs among gastric and colorectal cancers Egyptian patients. In order to achieve to that, 89 Biopsy and surgical specimens from the antrum and corpus mucosa of chronic gastritis subjects and gastric and colorectal carcinoma patients was collected for DNA extraction followed by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). The amplified PCR products of IL-1β-31C > T and IL-1β-511T > C were digested by incubation with the restriction endonuclease enzymes ALu1 and Ava1. Statistical analysis was carried out to determine the allele frequency distribution in the three studied groups. Also, the effect of the IL-1β -31 and -511 SNPs on nuclear factor binding was analyzed using Fluorescence Electrophoretic Mobility Shift Assay (EMSA), preceded by nuclear factor extraction from gastric and colorectal tissue samples and LPS stimulated monocytes. The results of this study showed that a significantly higher percentage of Egyptian gastric cancer patients have a homozygous CC genotype at the IL-1β-31 position and a heterozygous TC genotype at the IL-1β-511 position. Moreover, a significantly higher percentage of the colorectal cancer patients have a homozygous CC genotype at the IL-1β-31 and -511 positions as compared to the control group. In addition, the EMSA results showed that IL-1β-31C/T and IL-1β-511T/C SNPs do not affect nuclear factor binding. Results of this study suggest that the IL-1β-31 C/T and IL-1β-511 T/C may be correlated to the incidence of gastric cancer in Egyptian patients; however, similar findings couldn’t be proven in the colorectal cancer patients group for the IL-1β-511 T/C SNP. This is the first study to investigate IL-1β -31 and -511 SNPs in the Egyptian population.

Keywords: colorectal cancer, Egyptian patients, gastric cancer, interleukin-1β, single nucleotide polymorphisms

Procedia PDF Downloads 141

Authors: Deepak Loura

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Production and productivity of several crops in the country continue to be adversely affected by biotic (e.g., Insect-pests and diseases) and abiotic (e.g., water temperature and salinity) stresses. Over-dependence on pesticides and other chemicals is economically non-viable for the resource-poor farmers of our country. Further, pesticides can potentially affect human and environmental safety. While traditional breeding techniques and proper- management strategies continue to play a vital role in crop improvement, we need to judiciously use biotechnology approaches for the development of genetically modified crops addressing critical problems in the improvement of crop plants for sustainable agriculture. Modern biotechnology can help to increase crop production, reduce farming costs, and improve food quality and the safety of the environment. Genetic engineering is a new technology which allows plant breeders to produce plants with new gene combinations by genetic transformation of crop plants for improvement of agronomic traits. Advances in recombinant DNA technology have made it possible to have genes between widely divergent species to develop genetically modified or genetically engineered plants. Plant genetic engineering provides the strength to harness useful genes and alleles from indigenous microorganisms to enrich the gene pool for developing genetically modified (GM) crops that will have inbuilt (inherent) resistance to insect pests, diseases, and abiotic stresses. Plant biotechnology has made significant contributions in the past 20 years in the development of genetically engineered or genetically modified crops with multiple benefits. A variety of traits have been introduced in genetically engineered crops which include (i) herbicide resistance. (ii) pest resistance, (iii) viral resistance, (iv) slow ripening of fruits and vegetables, (v) fungal and bacterial resistance, (vi) abiotic stress tolerance (drought, salinity, temperature, flooding, etc.). (vii) quality improvement (starch, protein, and oil), (viii) value addition (vitamins, micro, and macro elements), (ix) pharmaceutical and therapeutic proteins, and (x) edible vaccines, etc. Multiple genes in transgenic crops can be useful in developing durable disease resistance and a broad insect-control spectrum and could lead to potential cost-saving advantages for farmers. The development of transgenic to produce high-value pharmaceuticals and the edible vaccine is also under progress, which requires much more research and development work before commercially viable products will be available. In addition, molecular-aided selection (MAS) is now routinely used to enhance the speed and precision of plant breeding. Newer technologies need to be developed and deployed for enhancing and sustaining agricultural productivity. There is a need to optimize the use of biotechnology in conjunction with conventional technologies to achieve higher productivity with fewer resources. Therefore, genetic modification/ engineering of crop plants assumes greater importance, which demands the development and adoption of newer technology for the genetic improvement of crops for increasing crop productivity.

Keywords: biotechnology, plant genetic engineering, genetically modified, biotic, abiotic, disease resistance

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Authors: Ali Bayram, Burak Uz, Remzi Yiğiter

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The impairment of lipid metabolism in the central nervous system has been suggested as a critical factor of Alzheimer’s disease (AD) pathogenesis. Homo sapiens acyl-coenyme A synthetase short-chain family member 2 (ACSS2) gene encodes the enzyme acetyl-Coenzyme A synthetase (AMP forming; AceCS) providing acetyl-coenzyme A (Ac-CoA) for various physiological processes, such as cholesterol and fatty acid synthesis, as well as the citric acid cycle. We investigated ACSS2, transcript variant 1 (ACSS2*1), mRNA levels in the peripheral blood mononuclear cells (PBMC) of patients with AD and compared them with the controls. The study group comprised 50 patients with the diagnosis of AD who have applied to Gaziantep University Faculty of Medicine, and Department of Neurology. 49 healthy individuals without any neurodegenerative disease are included as controls. ACSS2 mRNA expression in PBMC of AD/control patients was 0.495 (95% confidence interval: 0.410-0.598), p= .000000001902). Further studies are needed to better clarify this association.

Keywords: Alzheimer’s disease, ACSS2 Genes, mRNA expression, RT-PCR

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Authors: Sezin Ekşioğlu

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Multilabel classification has a huge importance for several applications, it is also a challenging research topic. It is a kind of supervised learning that contains binary targets. The distance between multilabel and binary classification is having more than one class in multilabel classification problems. Features can belong to one class or many classes. There exists a wide range of applications for multi label prediction such as image labeling, text categorization, gene functionality. Even though features are classified in many classes, they may not always be properly classified. There are many ensemble methods for the classification. However, most of the researchers have been concerned about better multilabel methods. Especially little ones focus on both efficiency of classifiers and pairwise relationships at the same time in order to implement better multilabel classification. In this paper, we worked on modified ensemble methods by getting benefit from k-Nearest Neighbors and neural network structure to address issues within a beneficial way and to get better impacts from the multilabel classification. Publicly available datasets (yeast, emotion, scene and birds) are performed to demonstrate the developed algorithm efficiency and the technique is measured by accuracy, F1 score and hamming loss metrics. Our algorithm boosts benchmarks for each datasets with different metrics.

Keywords: multilabel, classification, neural network, KNN

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Authors: Ilian Badjakov, Ivayla Dincheva, Violeta Kondakova, Rossitza Batchvarova

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In this study, we present our initial results on the assessment of genetic diversity among wild Bulgarian berry accessions (Rubus idaeus L. Fragaria Vesca L., Vaccinium vitis-idaea L., Vaccinium myrtillus L.) using Random Amplified Polymorphic DNA (RAPDs) markers. Leaves and fruits were collected from two natural habitats - the Balkan Mountain and the Mountain of Orpheus - Rhodope Mountain. All accessions were screened for their polymorphism using five RAPD primers. The phylogenetic distances calculated from RAPD data ranged from 0.29 to 0.82 thus indicating that a high level of gene diversity is present in the selected genotypes. In order to characterize further the structure and grouping of berry accessions, a dendrogram deriving from UPGMA cluster analysis based on the genetic similarity (GS) coefficient matrix was designed. RAPD analysis provided to be efficient for discrimination of accessions within the same species with similar morphological characters

Keywords: Bulgarian wild berries, genetic diversity, RAPD, UPGMA

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Authors: Zou Yihui

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The integration of the traditional landscape of the ancient city and the poet's emotions and symbolization into ancient poetry is the unique cultural gene and spiritual core of the historical city, and re-understanding the historical landscape pattern from the poetry is conducive to continuing the historical city context and improving the current situation of the gradual decline of the poetry of the modern historical urban landscape. Starting from Tang poetry uses semantic analysis methods、combined with text mining technology, entry mining, word frequency analysis, and cluster analysis of the landscape information of Tang Chang'an City were carried out, and the method framework for analyzing the urban landscape form based on poetry text was constructed. Nearly 160 poems describing the landscape of Tang Chang'an City were screened, and the poetic landscape characteristics of Tang Chang'an City were sorted out locally in order to combine with modern urban spatial development to continue the urban spatial context.

Keywords: Tang Chang'an City, poetic texts, semantic analysis, historical landscape

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Authors: Ihtisham Bukhari

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In the current study, we enrolled a 46, XY phenotypically female patient bearing testes in her inguinal canal. DNA sequencing of the AR gene detected a missense mutation C.1715A > G (p. Y572C) in exon 2 which is already known to cause Complete androgen insensitivity syndrome (CAIS). We further studied the effects of this mutation on the testicular histopathology of the patient. No spermatocytes were seen in the surface spreading of testicular tissues while H&E staining showed that seminiferous tubules predominantly have only Sertoli cells. To confirm this meiotic failure is likely due to the current AR mutation we performed mRNA expression of genes associated with AR pathway, expression and location of the associated proteins in testicular tissues. Western blot and real-time PCR data showed that the patient had high levels of expression of AMH, SOX9, and INNB in testis. Tubules were stained with SOX9 and AMH which revealed Sertoli cell maturation arrest. Therefore, we suggest that AR mutation enhances AMH expression which ultimately leads to failure in the maturation of Sertoli cells and failure in spermatogenesis.

Keywords: androgen receptor, spermatogenesis, infertility, Sertoli cell only syndrome

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Authors: M. F. Elzarei, A. M. Alseaf

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Hundred uncorrelated animal and thirty six markers were used in this study to study the molecular characterization of Saudi native Ardi goat (BM18189, ILSTS030, INRA005, OarFCB48, BM2113, ILSTS033, INRA023, RM088, CSRD247, ILSTS034, INRA063, SRCRSP1, ILSTS002, ILSTS044, INRA172, SRCRSP5, ILSTS005, ILSTS049, MAF70, SRCRSP8, ILSTS011, ILSTS058, OarAE54, SRCRSP9, ILSTS019, ILSTS059, OARCP34, TGLA53, ILSTS022, ILSTS082, OARE129, TGLA73, ILSTS029, ILSTS087, OARE193, and RM004). Ardi goat showed high variability. The mean number of alleles per locus ranged from 5 in SRCRSP1 locus to 13.5 in CSRD247 locus. Gene diversities varied within a wide range, from 0.53 in ILSTS002 locus to 0.86 in RM088 locus. Hardy-Weinberg equilibrium was tested in order to evaluate the significance of inbreeding occurring in each locus in Ardi population. Only SRCRSP9, INRA005, ILSTS030 loci showed significance in this way.

Keywords: molecular characterization, microsatellite markers, Ardi goats, Hardy-Weinberg equilibrium

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Authors: Ilknur Tuncer, Nihayet Bizsel

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The studies in bacterial diversity and antimicrobial resistance in coastal areas are important to understand the variability in the community structures and metabolic activities. In the present study, antimicrobial susceptibility and phylogenetic analysis of bacteria isolated from stations with different depths and influenced by terrestrial and marine fluxes in eastern Aegean Sea were illustrated. 51% of the isolates were found as resistant and 14% showed high MAR index indicating the high-risk sources of contamination in the environment. The resistance and the intermediate levels and high MAR index of the study area were 38–60%, 11–38% and 0–40%, respectively. According to 16S rRNA gene analysis, it was found that the isolates belonged to two phyla Firmicutes and Gammaproteobacteria with the genera Bacillus, Halomonas, Oceanobacillus, Photobacterium, Pseudoalteromonas, Psychrobacter, and Vibrio. 47% of Bacillus strains which were dominant among all isolates were resistant. In addition to phylogenetically diverse bacteria, the variability in resistance, intermediate and high MAR index levels of the study area indicated the effect of geographical differences.

Keywords: bacterial diversity, multiple antibiotic resistance, 16S rRNA genes, Aegean Sea

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Authors: Wen-Yang Hu, Ranli Lu, Mark Maienschein-Cline, Danping Hu, Larisa Nonn, Toshi Shioda, Gail S. Prins

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Background: Genetic mutations are highly associated with increased prostate cancer risk. In addition to whole genome sequencing, somatic mutations can be identified by aligning transcriptome sequences to the human genome. Here we analyzed bulk RNAseq and single cell RNAseq data of human prostate cancer cells and their matched non-cancer cells in benign regions from 4 individual patients. Methods: Sequencing raw reads were aligned to the reference genome hg38 using STAR. Variants were annotated using Annovar with respect to overlap gene annotation information, effect on gene and protein sequence, and SIFT annotation of nonsynonymous variant effect. We determined cancer-specific novel alleles by comparing variant calls in cancer cells to matched benign cells from the same individual by selecting unique alleles that were only detected in the cancer samples. Results: In bulk RNAseq data from 3 patients, the most common variants were the noncoding mutations at UTR3/UTR5, and the major variant types were single-nucleotide polymorphisms (SNP) including frameshift mutations. C>T transversion is the most frequently presented substitution of SNP. A total of 222 genes carrying unique exonic or UTR variants were revealed in cancer cells across 3 patients but not in benign cells. Among them, transcriptome levels of 7 genes (CITED2, YOD1, MCM4, HNRNPA2B1, KIF20B, DPYSL2, NR4A1) were significantly up or down regulated in cancer stem cells. Out of the 222 commonly mutated genes in cancer, 19 have nonsynonymous variants and 11 are damaged genes with variants including SIFT, frameshifts, stop gain/loss, and insertions/deletions (indels). Two damaged genes, activating transcription factor 6 (ATF6) and histone demethylase KDM3A are of particular interest; the former is a survival factor for certain cancer cells while the later positively activates androgen receptor target genes in prostate cancer. Further, single cell RNAseq data of cancer cells and their matched non-cancer benign cells from both primary 2D and 3D tumoroid cultures were analyzed. Similar to the bulk RNAseq data, single cell RNAseq in cancer demonstrated that the exonic mutations are less common than noncoding variants, with SNPs including frameshift mutations the most frequently presented types in cancer. Compared to cancer stem cell enriched-3D tumoroids, 2D cancer cells carried 3-times higher variants, 8-times more coding mutations and 10-times more nonsynonymous SNP. Finally, in both 2D primary and 3D tumoroid cultures, cancer stem cells exhibited fewer coding mutations and noncoding SNP or insertions/deletions than non-stem cancer cells. Summary: Our study demonstrates the usefulness of bulk and single cell RNAseaq data in identifying somatic mutations in prostate cancer, providing an alternative method in screening candidate genes for prostate cancer diagnosis and potential therapeutic targets. Cancer stem cells carry fewer somatic mutations than non-stem cancer cells due to their inherited immortal stand DNA from parental stem cells that explains their long-lived characteristics.

Keywords: prostate cancer, stem cell, genomic mutation, RNAseq

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Authors: Rashad Al-Hindi

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The aims of the study were to isolate and identify potential probiotic lactic acid bacteria due to their therapeutic and food preservation importance. Sixty-three suspected lactic acid bacteria (LAB) strains were isolated from thirteen different raw milk and fermented milk product samples of various animal origins manufactured indigenously in the Kingdom of Saudi Arabia using de Man, Rogosa and Sharpe (MRS) agar medium and various incubation conditions. The identification of forty-six selected LAB strains was performed using molecular methods (16S rDNA gene sequencing). The LAB counts in certain samples were higher under microaerobic incubation conditions than under anaerobic conditions. The identified LAB belonged to the following genera: Enterococcus (16 strains), Lactobacillus (9 strains), Weissella (10 strains), Streptococcus (8 strains) and Lactococcus (3 strains), constituting 34.78%, 19.57%, 21.74%, 17.39% and 6.52% of the suspected isolates, respectively. This study noted that the raw milk and traditional fermented milk products of Saudi Arabia, especially stirred yogurt (Laban) made from camel milk, could be rich in LAB. The obtained LAB strains in this study will be tested for their probiotic potentials in another ongoing study.

Keywords: dairy, LAB, probiotic, Saudi Arabia

Procedia PDF Downloads 287

Authors: Inna Kornienko, Svetlana Guryeva, Anatoly Shekhter, Elena Petersen

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Transplantation is an effective treatment option for patients suffering from different end-stage diseases. However, it is plagued by a constant shortage of donor organs and the subsequent need of a lifelong immunosuppressive therapy for the patient. Currently some researchers look towards using of pig organs to replace human organs for transplantation since the matrix derived from porcine organs is a convenient substitute for the human matrix. As an initial step to create a new ex vivo tissue engineered model, optimized protocols have been created to obtain organ-specific acellular matrices and evaluated their potential as tissue engineered scaffolds for culture of normal cells and tumor cell lines. These protocols include decellularization by perfusion in a bioreactor system and immersion-agitation on an orbital shaker with use of various detergents (SDS, Triton X-100) and freezing. Complete decellularization – in terms of residual DNA amount – is an important predictor of probability of immune rejection of materials of natural origin. However, the signs of cellular material may still remain within the matrix even after harsh decellularization protocols. In this regard, the matrices obtained from tissues of low-immunogenic pigs with α3Galactosyl-tranferase gene knock out (GalT-KO) may be a promising alternative to native animal sources. The research included a study of induced effect of frozen and fresh fragments of GalT-KO skin on healing of full-thickness plane wounds in 80 rats. Commercially available wound dressings (Ksenoderm, Hyamatrix and Alloderm) as well as allogenic skin were used as a positive control and untreated wounds were analyzed as a negative control. The results were evaluated on the 4th day after grafting, which corresponds to the time of start of normal wound epithelization. It has been shown that a non-specific immune response in models treated with GalT-Ko pig skin was milder than in all the control groups. Research has been performed to measure technical skin characteristics: stiffness and elasticity properties, corneometry, tevametry, and cutometry. These metrics enabled the evaluation of hydratation level, corneous layer husking level, as well as skin elasticity and micro- and macro-landscape. These preliminary data may contribute to development of personalized transplantable organs from GalT-Ko pigs with significantly limited potential of immune rejection. By applying growth factors to a decellularized skin sample it is possible to achieve various regenerative effects based on the particular situation. In this particular research BMP2 and Heparin-binding EGF-like growth factor have been used. Ideally, a bioengineered organ must be biocompatible, non-immunogenic and support cell growth. Porcine organs are attractive for xenotransplantation if severe immunologic concerns can be bypassed. The results indicate that genetically modified pig tissues with knock-outed α3Galactosyl-tranferase gene may be used for production of low-immunogenic matrix suitable for transplantation.

Keywords: decellularization, low-immunogenic, matrix, scaffolds, transplants

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Authors: Jai Myung Yang, Na Young Kim, Sung Ho Shin

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The adverse reactions of current live smallpox vaccines and potential use of smallpox as a bioterror weapon have heightened the development of new effective vaccine for this infectious disease. In the present study, DNA vaccine vector was produced which was optimized for expression of the vaccinia virus L1 antigen in the mouse model. A plasmid IgM-tL1R, which contains codon-optimized L1R gene, was constructed and fused with an IgM signal sequence under the regulation of a SV40 enhancer. The expression and secretion of recombinant L1 protein was confirmed in vitro 293 T cell. Mice were administered the DNA vaccine by electroporation and challenged with vaccinia virus. We observed that immunization with IgM-tL1R induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Isotyping studies reveal that immunoglobulin G2 (IgG2) antibody predominated after the immunization, indicative of a T helper type 1 response. Our results suggest that an optimized DNA vaccine, IgM-tL1R, can be effective in stimulating anti-vaccinia virus immune response and provide protection against lethal orthopoxvirus challenge.

Keywords: DNA vaccine, electroporation, L1R, vaccinia virus

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Authors: C. I. Boshoff, S. Steenkamp-Jonker

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Cystic echinococcosis (CE), caused by Echinococcus granulosus is an important parasitic infection in livestock worldwide, with severe zoonotic potential. It is important to understand the variability of Echinococcus granulosus, as genotype variations may influence lifecycle patterns, development rate, and transmission. Cystic Echinococcus samples were collected from domestic animals in Eastern Cape Province, South Africa. A molecular study was performed on 14 hydatid cysts obtained from caprine, ovine and bovine livers in order to determine the Echinococcus granulosus strain present in these hosts. The sequencing of the mitochondrial cytochrome C oxidase subunit I (coxI) gene of the hydatid cysts produced sequences of 400 bp for each sample analysed. These sequences were aligned with those present in GenBank and a phylogenetic tree was constructed. Based on coxI genotype the isolates could be grouped into E. granulosus sensu stricto. The findings of the study represent a pilot molecular study on Echinococcus from domestic animals undertaken in South Africa.

Keywords: Echinococcus granulosus, genotypes, livestock, South Africa

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Authors: Noshaba Dilbar, Aisha Tahir, Amer Jamil

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During the last decade, DNA barcoding proved to be an authentic tool for discovery and identification of plants. In the present study, DNA barcoding of two species, Fagonia arabica and Fagonia indica was done for differentiation by using matK region. matK gene is considered as a universal barcode because of its easy alignment and high discrimination ability. In this study, matK yielded 100% sequencing results. The sequences from both plants were aligned at clustal W and observed that there is no nucleotide variation and polymorphism among both sequences. This was further analysed by BLAST which showed the similar sequences from different plants belonging to same family but didn’t find sequence of both species. Considering this, the resulted sequence was submitted by the name of Fagonia arabica with accession number KM276890. In the end, we analysed the results from BOLD which gave us the final conclusion that both plants are same as their matK sequences are 100% identical. In literature, both Fagonia indica and Fagonia arabica names are used for this plant but there is no clear differentiation has been observed in these plants. Results evaluate that Fagonia indica and Fagonia arabica are the alternative names of same plant.

Keywords: DNA barcoding, Fagonia arabica, Fagonia indica, matK

Procedia PDF Downloads 153