Search results for: gene knockout

Authors: Fernanda Gonçalves, Karla Alcântara, Vanessa Moura, Patrícia Nolasco, Jorge Kalil, Maristela Hernandez

Abstract:

Introduction: Atherosclerosis is a chronic disease where two striking features are observed: retention of lipids and inflammation. Understanding the interaction between immune cells and lipoproteins involved in atherogenesis are urgent challenges, since cardiovascular diseases are the leading cause of death worldwide. Macrophages are critical to the development of atherosclerotic plaques and in the perpetuation of inflammation in these lesions. These cells are also directly involved in unstable plaque rupture. Recently different populations of macrophages are being identified in atherosclerotic lesions. Although the presence of M2 macrophages (macrophages activated by the alternative pathway, eg. The IL-4) has been identified, the function of these cells in atherosclerosis is not yet defined. M2 macrophages have a high endocytic capacity, they promote remodeling of tissues and to have anti-inflammatory activity. However, in atherosclerosis, especially unstable plaques, severe inflammatory reaction, accumulation of cellular debris and intense degradation of the tissue is observed. Thus, it is possible that the M2 macrophages have altered function (phenotype) in atherosclerosis. Objective: Our aim is to evaluate if the presence of oxidized LDL alters the phenotype and function of M2 macrophages in vitro. Methods: For this, we will evaluate whether the addition of lipoprotein in M2 macrophages differentiated in vitro with IL -4 induces 1) a reduction in the secretion of anti-inflammatory cytokines (CBA and ELISA), 2) secretion of inflammatory cytokines (CBA and ELISA), 3) expression of cell activation markers (Flow cytometry), 4) alteration in gene expression of molecules adhesion and extracellular matrix (Real-Time PCR) and 5) Matrix degradation (confocal microscopy). Results: In oxLDL stimulated M2 macrophages cultures we did not find any differences in the expression of the cell surface markers tested, including: HLA-DR, CD80, CD86, CD206, CD163 and CD36. Also, cultures stimulated with oxLDL had similar phagocytic capacity when compared to unstimulated cells. However, in the supernatant of these cultures an increase in the secretion of the pro-inflammatory cytokine IL-8 was detected. No significant changes where observed in IL-6, IL-10, IL-12 and IL-1b levels. The culture supernatant also induced massive extracellular matrix (produced by mouse embryo fibroblast) filaments degradation. When evaluating the expression of 84 extracellular matrix and adhesion molecules genes, we observed that the stimulation of oxLDL in M2 macrophages decreased 47% of the genes and increased the expression of only 3% of the genes. In particular we noted that oxLDL inhibit the expression of 60% of the genes constituents of extracellular matrix and collagen expressed by these cells, including fibronectin1 and collagen VI. We also observed a decrease in the expression of matrix protease inhibitors, such as TIMP 2. On the opposite, the matricellular protein thrombospondin had a 12 fold increase in gene expression. In the presence of native LDL 90% of the genes had no altered expression. Conclusion: M2 macrophages stimulated with oxLDL secrete the pro-inflammatory cytokine IL-8, have an altered extracellular matrix constituents gene expression, and promote the degradation of extracellular matrix. M2 macrophages may contribute to the perpetuation of inflammation in atherosclerosis and to plaque rupture.

Keywords: atherosclerosis, LDL, macrophages, m2

Procedia PDF Downloads 335

Authors: Ishani D. Goonasekara, Erio Camporesi, Timur Bulgakov, Rungtiwa Phookamsak, Kevin D. Hyde

Abstract:

Phaeosphaeriaceae comprises a large number of species occurring mainly on grasses and cereal crops as endophytes, saprobes and especially pathogens. Parastagonospora is an important genus in Phaeosphaeriaceae that includes pathogens causing leaf and glume blotch on cereal crops. Currently, there are fifteen Parastagonospora species described, including both pathogens and saprobes. In this study, one sexual morph species and an asexual morph species, occurring as saprobes on members of Poaceae are introduced based on morphology and a combined molecular analysis of the LSU, SSU, ITS, and RPB2 gene sequence data. The sexual morph species Parastagonospora elymi was isolated from a Russian sample of Elymus repens, a grass commonly known as couch grass, and important for grazing animals, as a weed and used in traditional Austrian medicine. P. elymi is similar to the sexual morph of P. avenae in having cylindrical asci, bearing 8, overlapping biseriate, fusiform ascospores but can be distinguished by its subglobose to conical shaped, wider ascomata. In addition, no sheath was observed surrounding the ascospores. The asexual morph species was isolated from a specimen from Italy, on Dactylis glomerata, a commonly found grass distributed in temperate regions. It is introduced as Parastagonospora macrouniseptata, a coelomycete, and bears a close resemblance to P. allouniseptata and P. uniseptata in having globose to subglobose, pycnidial conidiomata and hyaline, cylindrical, 1-septate conidia. However, the new species could be distinguished in having much larger conidiomata. In the phylogenetic analysis which consisted of a maximum likelihood and Bayesian analysis P. elymi showed low bootstrap support, but well segregated from other strains within the Parastagonospora clade. P. neoallouniseptata formed a sister clade with P. allouniseptata with high statistical support.

Keywords: dothideomycetes, multi-gene analysis, Poaceae, saprobes, taxonomy

Procedia PDF Downloads 118

Authors: Rasha Ahmadi

Abstract:

In December 2019, a coronavirus, currently identified as SARS-CoV-2, produced a series of acute atypical respiratory illnesses in Wuhan, Hubei Province, China. The sickness induced by this virus was named COVID-19. The virus is transmittable between humans and has caused pandemics worldwide. The number of death tolls continues to climb and a huge number of countries have been obliged to perform social isolation and lockdown. Lack of focused therapy continues to be a problem. Epidemiological research showed that senior patients were more susceptible to severe diseases, whereas children tend to have milder symptoms. In this study, we focus on other possible side effects of COVID-19 and more detailed treatment strategies. Using bioinformatics analysis, we first isolated the gene expression profile of patients with severe COVID-19 from the GEO database. Patients' blood samples were used in the GSE183071 dataset. We then categorized the genes with high and low expression. In the next step, we uploaded the genes separately to the Enrichr database and evaluated our data for signs and symptoms as well as related medication regimens. The results showed that 138 genes with high expression and 108 genes with low expression were observed differentially in the severe COVID-19 VS control group. Symptoms and diseases such as embolism and thrombosis of the abdominal aorta, ankylosing spondylitis, suicidal ideation or attempt, regional enteritis were observed in genes with high expression and in genes with low expression of acute and subacute forms of ischemic heart, CNS infection and poliomyelitis, synovitis and tenosynovitis. Following the detection of diseases and possible signs and symptoms, Carmustine, Bithionol, Leflunomide were evaluated more significantly for high-expression genes and Chlorambucil, Ifosfamide, Hydroxyurea, Bisphenol for low-expression genes. In general, examining the different and invisible aspects of COVID-19 and identifying possible treatments can help us significantly in the emergency and hospitalization of patients.

Keywords: phenotypes, drug regimens, gene expression profiles, bioinformatics analysis, severe COVID-19

Procedia PDF Downloads 142

Authors: Erfan Rahimi, Hadi Bahari Far, Mojgan Shikhpour

Abstract:

Background: Urinary tract infection is one of the most common nosocomial infections, especially among women. E. coli is one of the main causes of urinary tract infections and one of the most common antibiotics to fight this bacterium is ampicillin. As conventional antibiotics led to bacterial antibiotic resistance, modification of the pure drugs can address this issue. The aim of this study was to prepare nanofluids containing carbon nanotubes conjugated with ampicillin to improve drug performance and reduce antibiotic resistance. Methods: Multi-walled carbon nanotubes (MWCNTs) were activated with thionyl chloride by reflux system and nanofluids containing antibiotics were prepared by ultrasonic method. The properties of the prepared nano-drug were investigated by general element analysis, infrared spectroscopy, Raman spectroscopy, scanning electron microscopy and transmission electron microscopy. After the treatment of the desired strain with nanofluid, microbial studies were performed to evaluate the antibacterial effects and molecular studies were carried out to measure the expression of the resistance gene AcrAB. Result: We have shown that the antimicrobial effect of ampicillin-functionalized MWCNTs at low concentrations performed better than that of the conventional drug in both resistant and ATCC strains. Also, a decrease in antibiotic resistance of bacteria treated with ampicillin-functionalized MWCNTs compared to the pure drug was observed. Also, ampicillin-functionalized MWCNTs downregulated the expression of AcrAB in treated bacteria. Conclusion: Because carbon nanotubes are capable of destroying the bacterial wall, which provides antibiotic resistance features in bacteria, their usage in the form of nanofluids can make lower dosages (about three times less) than that of the pure drug more effective. Additionally, the expression of the bacterial resistance gene AcrAB decreased, thereby reducing antibiotic resistance and improving drug performance against bacteria.

Keywords: urinary tract infection, antibiotic resistance, carbon nanotube, nanofluid

Procedia PDF Downloads 146

Authors: Mathula Thangarajh

Abstract:

Duchenne muscular dystrophy (DMD) is a severe form of X-linked muscular dystrophy caused by mutations in the dystrophin gene resulting in progressive skeletal muscle weakness. Boys with DMD also have significant cognitive disabilities. The intelligence quotient of boys with DMD, compared to peers, is approximately one standard deviation below average. Detailed neuropsychological testing has demonstrated that boys with DMD have a global developmental impairment, with verbal memory and visuospatial skills most significantly affected. Furthermore, the total brain volume and gray matter volume are lower in children with DMD compared to age-matched controls. These results are suggestive of a significant structural and functional compromise to the developing brain as a result of absent dystrophin protein expression. There is also some genetic evidence to suggest that mutations in the 3’ end of the DMD gene are associated with more severe neurocognitive problems. Our working hypothesis is that (i) boys with DMD do not make gains in neurodevelopmental skills compared to typically developing children and (ii) women carriers of DMD mutations may have subclinical cognitive deficits. We also hypothesize that there may be an intergenerational vulnerability of cognition, with boys of DMD-carrier mothers being more affected cognitively than boys of non-DMD-carrier mothers. The objectives of this study are: 1. Assess the neurodevelopment in boys with DMD at 4-time points and perform baseline neuroradiological assessment, 2. Assess cognition in biological mothers of DMD participants at baseline, 3. Assess possible correlation between DMD mutation and cognitive measures. This study also explores functional brain abnormalities in people with DMD by exploring how regional and global connectivity of the brain underlies executive function deficits in DMD. Such research can contribute to a better holistic understanding of the cognition alterations due to DMD and could potentially allow clinicians to create better-tailored treatment plans for the DMD population. There are four study visits for each participant (baseline, 2-4 weeks, 1 year, 18 months). At each visit, the participant completes the NIH Toolbox Cognition Battery, a validated psychometric measure that is recommended by NIH Common Data Elements for use in DMD. Visits 1, 3, and 4 also involve the administration of the BRIEF-2, ABAS-3, PROMIS/NeuroQoL, PedsQL Neuromuscular module 3.0, Draw a Clock Test, and an optional fMRI scan with the N-back matching task. We expect to enroll 52 children with DMD, 52 mothers of children with DMD, and 30 healthy control boys. This study began in 2020 during the height of the COVID-19 pandemic. Due to this, there were subsequent delays in recruitment because of travel restrictions. However, we have persevered and continued to recruit new participants for the study. We partnered with the Muscular Dystrophy Association (MDA) and helped advertise the study to interested families. Since then, we have had families from across the country contact us about their interest in the study. We plan to continue to enroll a diverse population of DMD participants to contribute toward a better understanding of Duchenne Muscular Dystrophy.

Keywords: neurology, Duchenne muscular dystrophy, muscular dystrophy, cognition, neurodevelopment, x-linked disorder, DMD, DMD gene

Procedia PDF Downloads 99

Authors: Adel Alblihy, Reem Ali, Mashael Algethami, Ahmed Shoqafi, Michael S. Toss, Juliette Brownlie, Natalie J. Tatum, Ian Hickson, Paloma Ordonez Moran, Anna Grabowska, Jennie N. Jeyapalan, Nigel P. Mongan, Emad A. Rakha, Srinivasan Madhusudan

Abstract:

Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n=331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p=0.002). In the ovarian cancer genome atlas (TCGA) cohort (n=498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p<0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n=1259), Mre11 overexpression was associated with poor PFS (p=0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer.

Keywords: MRE11; XRCC1, ovarian cancer, platinum sensitization, synthetic lethality

Procedia PDF Downloads 129

Authors: Stéfani T. A. Dantas, Laura T. S. Takume, Bruna F. Rossi, Érika R. Bonsaglia, Ivana G. Castilho, José C. F. Pantoja, Ary Fernandes Júnior, Juliano L. Gonçalves, Marcos V. Santos, Rinaldo A. Mota, Vera L. M. Rall

Abstract:

Staphylococcus aureus is one of the main pathogens causing contagious bovine mastitis, being more frequently isolated from subclinical form, although the clinical form also occurs. Clinical mastitis cause visual signs, such as swelling, fever, hardening of the mammary gland, or any change in the characteristics of the milk. Considering the subclinical type, there are no visible signs in the animal nor changes in the milk. S. aureus has many important virulence factors for the establishment of its pathogenicity in animals, such as enterotoxins, which are also responsible for foodborne poisoning. Our objective is to perform a comparative analysis between 103 isolates of S. aureus, obtained from the milk of cows with clinical mastitis and 103 more, from subclinical type, in relation to the presence of these enterotoxins and verify if their presence plays an important role in the signs of illness. We will investigate all enterotoxins described till now, such as sea-see, seg-sez, sel26, sel 27, se01, and se02 (This study was approved by the Sao Paulo State University Animal Use Ethics Committee, No. 0136/2017). For the PCR assay, we used Illustra Bacteria Mini Spin Kit for bacterial DNA. At this moment, we have already tested sea-see, seg-ser, sew, and sex, and the results have already been submitted to Fisher Exact Probability Test or Chi-square Test. Considering the isolates obtained from clinical mastitis, the most frequent enterotoxins were selw (99%), selx (78%) and selh (50.5%), and sec, see, sej, sell, selp,and ser were absent. Among the subclinics, selw (82.5%) selm (15.5%) and selx (14.6%) were the most frequent, and sea-see, seg, sei-sel, sem-ser were absent. We have already observed statistically significant differences for seb, seg, seh, sei, selo, selu, selw and selx. Other interesting results were the low number of genes in each isolate from subclinical mastitis [0 genes: 14 (13.6%); 1 gene: 55 (53.4%); 2 genes: 33 (32%) or 3: 1 (0.97%)] compared to clinical isolates [1 gene: 5 (4.9%); 2 genes: 29 (28.1%); 3 genes: 38 (36.9%); 4 genes: 14 (13.6%); 5 genes: 5 (4.9%); 6 genes: 4 (3.9%); 7 genes: 5 (4.9%); 8 genes: 2 (1.9%) and 9 genes: 1 (1%)]. Based on these results, we can conclude that enterotoxins indeed play an important role in clinical signs in cattle with mastitis.

Keywords: mastitis, S. aureus, PCR, staphylococcal enterotoxin

Procedia PDF Downloads 113

Authors: F. Saleh, F. Lyall, A. Abdulsid, L. Marks

Abstract:

Background: Labour in human is a complex biological process that involves interactions of neurological, hormonal and inflammatory pathways, with the placenta being a key regulator of these pathways. It is known that uterine contractions and labour pain cause physiological changes in gene expression in maternal and fetal blood, and in placenta during labour. Oxidative and inflammatory stress pathways are implicated in labour and they may cause alteration of placental gene expression. Additionally, in placental tissues, labour increases the expression of genes involved in placental oxidative stress, inflammatory cytokines, angiogenic regulators and apoptosis. Recently, Hydrogen Sulphide (H2S) has been considered as an endogenous gaseous mediator which promotes vasodilation and exhibits cytoprotective anti-inflammatory properties. The endogenous H2S is synthesised predominantly by two enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). As the H2S pathway has anti-oxidative and anti-inflammatory characteristics thus, we hypothesised that the expression of CBS and CSE in placental tissues would alter during labour. Methods: CBS and CSE expressions were examined in placentas using western blotting and RT-PCR in inner, middle and outer placental zones in placentas obtained from healthy non labouring women who delivered by caesarian section. These were compared with the equivalent zone of placentas obtained from women who had uncomplicated labour and delivered vaginally. Results: No differences in CBS and CSE mRNA or protein levels were found between the different sites within placentas in either the labour or non-labour group. There were no significant differences in either CBS or CSE expression between the two groups at the inner site and middle site. However, at the outer site there was a highly significant decrease in CBS protein expression in the labour group when compared to the non-labour group (p = 0.002). Conclusion: To the best of author’s knowledge, this is the first report to suggest that, CBS is expressed in a spatial manner within the human placenta. Further work is needed to clarify the precise function and mechanism of this spatial regulation although it is likely that inflammatory pathways regulation is a complex process in which this plays a role.

Keywords: anti-inflammatory, hydrogen sulphide, labour, oxidative stress

Procedia PDF Downloads 241

Authors: Sobia Shafqat, Saeed Ahmad Qaisrani

Abstract:

MicroRNAs play an essential role in the regulation and development of all processes in most eukaryotes because of their prospective part as mediators controlling cell growth and differentiation towards the exact position of RNAs response in plants under biotic and abiotic factors or stressors. In a few cases, Zn is oblivious poisonous for plants due to its heavy metal status. Some other metals are extremely toxic, like Cd, Hg, and Pb, but these elements require in rice for the programming of genes under abiotic stress resembling Zn stress when micro RNAs268 was importantly introduced in rice. The micro RNAs overexpressed in transgenic plants with an accumulation of a large amount of melanin dialdehyde, hydrogen peroxide, and an excessive quantity of Zn in the seedlings stage. Let out results for rice pliability under Zn stress micro RNAs act as negative controllers. But the role of micro RNA268 act as a modulator in different ecological condition. It has been explained clearly with a long understanding of the role of micro RNA268 under stress conditions; pliability and practically showed outcome to increase plant sufferance under Zn stress because micro RNAs is an intervention technique for gene regulation in gene expression. The proposed study was experimented with by using genetic factors of Zn stress and toxicity effect on rice plants done at District Vehari, Pakistan. The trial was performed randomly with three replications in a complete block design (RCBD). These blocks were controlled with different concentrations of genetic factors. By overexpression of micro RNA268 rice, seedling growth was not stopped under Zn deficiency due to the accumulation of a large amount of melanin dialdehyde, hydrogen peroxide, and an excessive quantity of Zn in their seedlings. Results showed that micro RNA268 act as a negative controller under Zn stress. In the end, under stress conditions, micro RNA268 showed the necessary function in the tolerance of rice plants. The directorial work sketch gave out high agronomic applications and yield outcomes in rice with a specific amount of Zn application.

Keywords: micro RNA268, zinc, rice, agronomic approach

Procedia PDF Downloads 61

Authors: Zhala Ahmad, Zainab Lazim, Haider Hamzah

Abstract:

Bacterial resistance is a pressing global health issue, with multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) strains to pose a serious threat. In this context, researchers are investigating effective, safe, and affordable metabolites to combat these pathogens. This study focuses on gum essential oil (GEO) extracted from Pistacia atlantica and its activity and the mechanism of action against XDR Gram-negative Acinetobacter baumannii. GEO was extracted by hydrodistillation and analyzed using GC-MS. Eleven A. baumannii isolates were collected from the ward environment of Burn and Plastic Surgery Hospital in Al Sulaymaniyah City, Iraq. They were identified using the VITEK 2 system, 16S rRNA gene, and confirmed with the blaₒₓₐ₋₅₁ gene; A. baumannii ATCC 19606 was used as a reference strain. The isolates were identified as resistant to twelve different antibiotics spanning six distinct antibiotic classes while showing susceptibility to tetracycline and trimethoprim. Over 40 chemical constituents were detected in the gum's essential oils, with α-pinene being the most abundant. GEO was found to inhibit the growth of A. baumannii isolates; the minimum inhibitory concentration (MIC) of GEO was 2.5 µl/ml. GEO induced protein leakage, phosphate, and potassium ion efflux, distorted cell morphology, and cell death in the tested bacteria. GEO exhibited bacterial clearance and anti-adhesion activity using Band-Aids. This study's findings suggest that GEO could be used as a potential alternative treatment for infectious diseases caused by XRD pathogens, shedding further light on the importance of GEO in biomedical applications. Future studies must focus on generating clinically feasible sources of GEO for testing in small animal models before proceeding to human trials, ensuring safe and effective translation from the laboratory to the clinic.

Keywords: antibiotic resistance, Acinetobacter baumannii, essential oils, Pistacia atlantica, alpha-pinene

Procedia PDF Downloads 71

Authors: Leixuri Aguirre, Iñaki Milton-Laskibar, Elizabeth Hijona, Luis Bujanda, Agnes M. Rimando, Maria P. Portillo

Abstract:

Introduction: In recent years great attention has been paid by scientific community to phenolic compounds as active biomolecules naturally present in foodstuffs due to their beneficial effects on health. Pterostilbene is a resveratrol dimethylether derivative which shows higher biodisponibility. Objective. To analyze the effects of two doses of pterostilbene on several markers of thermogenic capacity in a model of genetic obesity, which shows reduced thermogenesis. Methods: The experiment was conducted with thirty Zucker (fa/fa) rats that were distributed in 3 experimental groups, the control group and two groups orally administered with pterostilbene at 15 and 30 mg/kg body weight/day for 6 weeks. Gene expression of Ucp1, Pgc-1α, Cpt1b, Pparα, Nfr1, Tfam and Cox-2 were assessed by RT-PCR, protein expression of UCP1 and GLUT4 by western blot and enzyme activity of carnitine palmitoyl transferase 1b and citrate synthase by spectrophotometry in interscapular brown adipose tissue (iBAT). Statistical analysis was performed by using one way ANOVA and Newman-Keuls as post-hoc test. Results: Pterostilbene did not change gene expression of Pgc-1α. However, significant increases were found in the expression of Ucp1, Pparα, Nfr-1 and Cox-2. Protein expression of UCP1 and GLUT4 was increased in animals treated with pterostilbene, as well as the activities of CPT-1b and CS. These effects were observed with both doses of pterostilbene, without differences between them. Conclusions: These results show that pterostilbene increases thermogenic and oxidative capacity of brown adipose tissue in obese rats. Whether these effects effectively contribute to the anti-obesity properties of these compound needs further research. Acknowledgments: MINECO-FEDER (AGL2015-65719-R), Basque Government (IT-572-13), University of the Basque Country (ELDUNANOTEK UFI11/32), Institut of Health Carlos III (CIBERobn). Iñaki Milton is a fellowship from the Basque Government.

Keywords: brown adipose tissue, pterostilbene, thermogenesis, uncoupling protein 1

Procedia PDF Downloads 297

Authors: Roxane A. Legaie, Kjiana E. Schwab, Caroline E. Gargett

Abstract:

Studies looking at the changes in gene expression from RNAseq data often make use of linear models. It is also common practice to focus on a subset of data for a comparison of interest, leaving aside the samples not involved in this particular comparison. This work shows the importance of including all observations in the modeling process to better estimate variance parameters, even when the samples included are not directly used in the comparison under test. The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. The mesenchymal stem cells (MSCs) present in the endometrium are likely responsible for this remarkable regenerative capacity. However recent studies suggest that MSCs also plays a role in the pathogenesis of endometriosis, one of the most common medical conditions affecting the lower abdomen in women in which the endometrial tissue grows outside the womb. In this study we compared gene expression profiles between MSCs and non-stem cell counterparts (‘non-MSC’) obtained from women with (‘E’) or without (‘noE’) endometriosis from RNAseq. Raw read counts were used for differential expression analysis using a linear model with the limma-voom R package, including either all samples in the study or only the samples belonging to the subset of interest (e.g. for the comparison ‘E vs noE in MSC cells’, including only MSC samples from E and noE patients but not the non-MSC ones). Using the full dataset we identified about 100 differentially expressed (DE) genes between E and noE samples in MSC samples (adj.p-val < 0.05 and |logFC|>1) while only 9 DE genes were identified when using only the subset of data (MSC samples only). Important genes known to be involved in endometriosis such as KLF9 and RND3 were missed in the latter case. When looking at the MSC vs non-MSC cells comparison, the linear model including all samples identified 260 genes for noE samples (including the stem cell marker SUSD2) while the subset analysis did not identify any DE genes. When looking at E samples, 12 genes were identified with the first approach and only 1 with the subset approach. Although the stem cell marker RGS5 was found in both cases, the subset test missed important genes involved in stem cell differentiation such as NOTCH3 and other potentially related genes to be used for further investigation and pathway analysis.

Keywords: differential expression, endometriosis, linear model, RNAseq

Procedia PDF Downloads 432

Authors: Mehdi Soula, Yosra Mani, Estelle Saras, Antoine Drapeau, Raoudha Grami, Mahjoub Aouni, Jean-Yves Madec, Marisa Haenni, Wejdene Mansour

Abstract:

Multi-resistance to antibiotics in gram-negative bacilli and particularly in enterobacteriaceae, has become frequent in hospitals in Tunisia. However, data on antibiotic resistant bacteria in aquatic products are scarce. The aims of this study are to estimate the proportion of ESBL- and carbapenemase-producing Enterobacterales in seafood (clams and fish) in Tunisia and to molecularly characterize the collected isolates. Two types of seafood were sampled in unrelated markets in four different regions in Tunisia (641 pieces of farmed fish and 1075 mediterranean clams divided into 215 pools, and each pool contained 5 pieces). Once purchased, all samples were incubated in tubes containing peptone salt broth for 24 to 48h at 37°C. After incubation, overnight cultures were isolated on selective MacConkey agar plates supplemented with either imipenem or cefotaxime, identified using API20E test strips (bioMérieux, Marcy-l’Étoile, France) and confirmed by Maldi-TOF MS. Antimicrobial susceptibility was determined by the disk diffusion method on Mueller-Hinton agar plates and results were interpreted according to CA-SFM 2021. ESBL-producing Enterobacterales were detected using the Double Disc Synergy Test (DDST). Carbapenem-resistance was detected using an ertapenem disk and was respectively confirmed using the ROSCO KPC/MBL and OXA-48 Confirm Kit (ROSCO Diagnostica, Taastrup, Denmark). DNA was extracted using a NucleoSpin Microbial DNA extraction kit (Macherey-Nagel, Hoerdt, France), according to the manufacturer’s instructions. Resistance genes were determined using the CGE online tools. The replicon content and plasmid formula were identified from the WGS data using PlasmidFinder 2.0.1 and pMLST 2.0. From farmed fishes, nine ESBL-producing strains (9/641, 1.4%) were isolated, which were identified as E. coli (n=6) and K. pneumoniae (n=3). Among the 215 pools of 5 clams analyzed, 18 ESBL-producing isolates were identified, including 14 E. coli and 4 K. pneumoniae. A low isolation rate of ESBL-producing Enterobacterales was detected 1.6% (18/1075) in clam pools. In fish, the ESBL phenotype was due to the presence of the blaCTX-M-15 gene in all nine isolates, but no carbapenemase gene was identified. In clams, the predominant ESBL phenotype was blaCTX-M-1 (n=6/18). blaCPE (NDM1, OXA48) was detected only in 3 isolates ‘K. pneumoniae isolates’. Replicon typing on the strains carring the ESBL and carbapenemase gene revelead that the major type plasmid carried ESBL were IncF (42.3%) [n=11/26]. In all, our results suggest that seafood can be a reservoir of multi-drug resistant bacteria, most probably of human origin but also by the selection pressure of antibiotic. Our findings raise concerns that seafood bought for consumption may serve as potential reservoirs of AMR genes and pose serious threat to public health.

Keywords: BLSE, carbapenemase, enterobacterales, tunisian seafood

Procedia PDF Downloads 108

Authors: Akhil Raj Pushparajan, Ranjit Ramachandran, Jijimole Gopi Reji, Ajay Kumar Ramakrishnan

Abstract:

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death by an infectious disease. In spite of the availability of effective drugs and a vaccine, TB is a major health concern and was declared a global emergency by the World Health Organization (WHO). The success of intracellular pathogens like Mtb depends on its ability to overcome the challenging environment in the host. Gene regulation controlled by transcriptional regulators (TRs) plays a crucial role for the bacteria to adapt to the host environment. In vitro studies on gene regulatory mechanisms during dormancy and reactivation have provided insights into the adaptations employed by Mtb to survive in the host. Here we present our efforts to functionally characterize Rv1019, a putative TR of Mtb H37Rv which was found to be present at significantly varying levels during dormancy and reactivation in vitro. The expression of this protein in the dormancy-reactivation model was validated by qRT-PCR and western blot. By DNA- protein interaction studies and reporter assays we found that under normal laboratory conditions of growth this protein behaves as an auto-repressor and tetracycline was found to abrogate this repression by interfering with its ability to bind DNA. Further, by cDNA analysis, we found that this TR is co-transcribed with its downstream genes Rv1020 (mfd) and Rv1021 (mazG) which are involved in DNA damage response in Mtb. Constitutive expression of this regulator in the surrogate host M. smegmatis showed downregulation of the orthologues of downstream genes suggested that Rv1019 could negatively regulate these genes. Our finds also show that M. smegmatis expressing Rv1019 is sensitive to DNA damage suggests the role of this protein in regulating DNA damage response induced by oxidative stress. Because of its role in regulating DNA damage response which may help in the persistence of Mtb, Rv1019 could be used as a prospective target for therapeutic intervention to fight TB.

Keywords: auto-repressor, DNA repair, mycobacterium smegmatis, mycobacterium tuberculosis, tuberculosis

Procedia PDF Downloads 139

Authors: Sadaf Moeez, Madiha Khalid, Zoya Khalid, Sania Shaheen, Sumbul Khalid

Abstract:

Background: Inter-individual variation in response to metformin, which has been considered as a first line therapy for T2DM treatment is considerable. In the current study, it was aimed to investigate the impact of two genetic variants Leu125Phe (rs77474263) and Gly64Asp (rs77630697) in gene SLC47A1 on the clinical efficacy of metformin in T2DM Pakistani patients. Methods: The study included 800 T2DM patients (400 metformin responders and 400 metformin non-responders) along with 400 ethnically matched healthy individuals. The genotypes were determined by allele-specific polymerase chain reaction. In-silico analysis was done to confirm the effect of the two SNPs on the structure of genes. Association was statistically determined using SPSS software. Results: Minor allele frequency for rs77474263 and rs77630697 was 0.13 and 0.12. For SLC47A1 rs77474263 the homozygotes of one mutant allele ‘T’ (CT) of rs77474263 variant were fewer in metformin responders than metformin non-responders (29.2% vs. 35.5 %). Likewise, the efficacy was further reduced (7.2% vs. 4.0 %) in homozygotes of two copies of ‘T’ allele (TT). Remarkably, T2DM cases with two copies of allele ‘C’ (CC) had 2.11 times more probability to respond towards metformin monotherapy. For SLC47A1 rs77630697 the homozygotes of one mutant allele ‘A’ (GA) of rs77630697 variant were fewer in metformin responders than metformin non-responders (33.5% vs. 43.0 %). Likewise, the efficacy was further reduced (8.5% vs. 4.5%) in homozygotes of two copies of ‘A’ allele (AA). Remarkably, T2DM cases with two copies of allele ‘G’ (GG) had 2.41 times more probability to respond towards metformin monotherapy. In-silico analysis revealed that these two variants affect the structure and stability of their corresponding proteins. Conclusion: The present data suggest that SLC47A1 Leu125Phe (rs77474263) and Gly64Asp (rs77630697) polymorphisms were associated with the therapeutic response of metformin in T2DM patients of Pakistan.

Keywords: diabetes, T2DM, SLC47A1, Pakistan, polymorphism

Procedia PDF Downloads 159

Authors: Thomais Tsoulia, Arvind Y. M. Sundaram, Stine Braaen, Øyvind Haugland, Espen Rimstad, Øystein Wessel, Maria K. Dahle

Abstract:

Fish red blood cells (RBC) are nucleated, and in addition to their function in gas exchange, they have been characterized as mediators of immune responses. Salmonid RBC are the major target cells of Piscineorthoreovirus (PRV), a virus associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon. The activation of antiviral response genesin RBChas previously been described in ex vivo and in vivo PRV-infection models, but not explored in the initial virus encounter phase. In the present study, mRNA transcriptome responses were explored in erythrocytes from individual fish, kept ex vivo, and exposed to purified PRV for 24 hours. The responses were compared to responses in macrophage-like salmon head kidney (SHK-1) and endothelial-like Atlantic salmon kidney (ASK) cells, none of which support PRV replication. The comparative analysis showed that the antiviral response to PRV was strongest in the SHK-1 cells, with a set of 80 significantly induced genes (≥ 2-fold upregulation). In RBC, 46 genes were significantly upregulated, while ASK cells were not significantly responsive. In particular, the transcriptome analysis of RBC revealed that PRV significantly induced interferon regulatory factor 1 (IRF1) and interferon-induced protein with tetratricopeptide repeats 5-like (IFIT9). However, several interferon-regulated antiviral genes which have previously been reported upregulated in PRV infected RBC in vivo (myxovirus resistance (Mx), interferon-stimulated gene 15 (ISG15), toll-like receptor 3 (TLR3)), were not significantly induced after 24h of virus stimulation. In contrast to RBC, these antiviral response genes were significantly upregulated in SHK-1. These results confirm that RBC are involved in the innate immune response to viruses, but with a delayed antiviral response compared to SHK-1. A notable difference is that interferon regulatory factor 1 (IRF-1) is the most strongly induced gene in RBC, but not among the significantly induced genes in SHK-1. Putative differences in the binding, recognition, and response to PRV, and any link to effects on the ability of PRV to replicate remains to be explored.

Keywords: antiviral responses, atlantic salmon, piscine orthoreovirus-1, red blood cells, RNA-seq

Procedia PDF Downloads 189

Authors: Vera Lukasova, Eva Filova, Jana Dankova, Vera Sovkova, Matej Daniel, Michala Rampichova

Abstract:

Proper bone implants should promote fast adhesion of cells, stimulate cell differentiation and support the formation of bone tissue. Nowadays titanium is used as a biocompatible material capable of bone tissue integration. This study was focused on comparison of bioactive properties of two titanium alloys - beta titanium alloy Ti36Nb6Ta and standard medical titanium alloy Ti6A14V. The advantage of beta titanium alloy Ti36Nb6Ta is mainly that this material does not contain adverse elements like vanadium or aluminium. Titanium alloys were sterilized in ethanol, placed into 48 well plates and seeded with porcine mesenchymal stem cells. Cells were cultivated for 14 days in standard growth cultivation media with osteogenic supplements. Cell metabolic activity was quantified using MTS assay (Promega). Cell adhesion on day 1 and cell proliferation on further days were verified immunohistochemically using beta-actin monoclonal antibody and secondary antibody conjugated with AlexaFluor®488. Differentiation of cells was evaluated using alkaline phosphatase assay. Additionally, gene expression of collagen I was measured by qRT-PCR. Porcine mesenchymal stem cells adhered and spread well on beta titanium alloy Ti36Nb6Ta on day 1. During the 14 days’ time period the cells were spread confluently on the surface of the beta titanium alloy Ti36Nb6Ta. The metabolic activity of cells increased during the whole cultivation period. In comparison to standard medical titanium alloy Ti6A14V, we did not observe any differences. Moreover, the expression of collagen I gene revealed no statistical differences between both titanium alloys. Therefore, a beta titanium alloy Ti36Nb6Ta promotes cell adhesion, metabolic activity, proliferation and collagen I expression equally to standard medical titanium alloy Ti6A14V. Thus, beta titanium is a suitable material that provides sufficient biocompatible properties. This project was supported by the Czech Science Foundation: grant No. 16-14758S.

Keywords: beta titanium alloy, biocompatibility, differentiation, mesenchymal stem cells

Procedia PDF Downloads 494

Authors: Achitphol Chookaew, Paramee Thongsukhsai, Patamarerk Engsontia, Narongwit Nakwan, Pritsana Raugrut

Abstract:

Lung cancer is one of the most common malignancies and primary cause of death due to cancer worldwide. Non-small cell lung cancer (NSCLC) is the main subtype in which majority of patients present with advanced-stage disease. Herein, we analyzed differentially expressed genes to find potential biomarkers for lung cancer diagnosis as well as prognostic markers. We used transcriptome data from our 2 NSCLC patients and public data (GSE81089) composing of 8 NSCLC and 10 normal lung tissues. Differentially expressed genes (DEGs) between NSCLC and normal tissue and between early-stage and late-stage NSCLC were analyzed by the DESeq2. Pairwise correlation was used to find the DEGs with false discovery rate (FDR) adjusted p-value £ 0.05 and |log2 fold change| ³ 4 for NSCLC versus normal and FDR adjusted p-value £ 0.05 with |log2 fold change| ³ 2 for early versus late-stage NSCLC. Bioinformatic tools were used for functional and pathway analysis. Moreover, the top ten genes in each comparison group were verified the expression and survival analysis via GEPIA. We found 150 up-regulated and 45 down-regulated genes in NSCLC compared to normal tissues. Many immnunoglobulin-related genes e.g., IGHV4-4, IGHV5-10-1, IGHV4-31, IGHV4-61, and IGHV1-69D were significantly up-regulated. 22 genes were up-regulated, and five genes were down-regulated in late-stage compared to early-stage NSCLC. The top five DEGs genes were KRT6B, SPRR1A, KRT13, KRT6A and KRT5. Keratin 6B (KRT6B) was the most significantly increased gene in the late-stage NSCLC. From GEPIA analysis, we concluded that IGHV4-31 and IGKV1-9 might be used as diagnostic biomarkers, while KRT6B and KRT6A might be used as prognostic biomarkers. However, further clinical validation is needed.

Keywords: differentially expressed genes, early and late-stages, gene ontology, non-small cell lung cancer transcriptomics

Procedia PDF Downloads 114

Authors: H. Alijani, M. Jabari, S. Matroodi, H. Zolqarnein, A. Sharafi, I. Zamani

Abstract:

Actinomycetes, Gram-positive bacteria, are interesting as a main producer of secondary metabolites and are important industrially and pharmaceutically. The marine environment is a potential source for new actinomycetes, which can provide novel bioactive compounds and industrially important enzymes. The aims of this study were to isolate and identify novel actinomycetes from Persian Gulf sediments and screen these isolates for the production of secondary metabolites, especially antibiotics, Using phylogenetic (16S rRNA gene sequence), morphological and biochemical analyses. 15 different actinomycete strains from Persian Gulf sediments at a depth of 5-10 m were identified. DNA extraction was done using Cinnapure DNA Kit. PCR amplification of 16S rDNA gene was performed using F27 and R1492 primers. Phylogenetic tree analysis was performed using the MEGA 6 software. Most of the isolated strains belong to the genus namely Streptomyces (14), followed by Nocardiopsis (1). Antibacterial assay of the isolates supernatant was performed using a standard disc diffusion assay with replication (n=3). The results of disk diffusion assay showed that most active strain against Proteus volgaris and Bacillus cereus was AMJ1 (16.46±0.2mm and 13.78±0.2mm, respectively), against Salmonella sp. AMJ7 was the most effective strain (10.13±0.2mm), and AMJ1 and AHA5 showed more inhibitory activity against Escherichia coli (8.04±0.02 mm and 8.2±0.03 ). The AMJ6 strain showed best antibacterial activity against Klebsiella sp. (8.03±0.02mm). Antifungal activity of AMJ2 showed that it was most active strain against complex (16.05±0.02mm) and against Aspergillus flavus strain AMJ1 was most active strain (16.4±0.2mm) and highest antifungal activity against Trichophyton mentagrophytes, Microsporum gyp serum and Candida albicans, were shown by AHA1 (21.03±0.02mm), AHA3 and AHA7 (18±0.03mm), AMJ6 (21.03±0.2mm) respectively. Our results revealed that the marine actinomycetes of Persian Gulf sediments were potent source of novel antibiotics and bioactive compounds and indicated that the antimicrobial metabolites were extracellular. Most of the secondary metabolites and antibiotics are extracellular in nature and extracellular products of actinomycetes show potent antimicrobial activities.

Keywords: antibacterial activity, antifungal activity, marine actinomycetes, Persian Gulf

Procedia PDF Downloads 297

Authors: Javed Sidiqi, Stephen Baezinger, Stephen Wegulo

Abstract:

Wheat (Triticum aestivum L.) production continues to be challenged by foliar fungal diseases although significant improvement has been made to manage the diseases through developing resistant varieties and the fungicide use to ensure sufficient wheat is produced to meet the growing population’s need. Significant crop losses have been recorded in the history of grain production and yield losses due to fungal diseases, and the trend continues to threat food security in the world and particularly in the less developed countries. The impact of individual fungal diseases on grain yield has been studied extensively to determine crop losses. However, there is limited research available to find out the combined effects of fungal diseases on grain yield and the ways to effectively manage the diseases. Therefore, the objectives of this research were to study the effect of fungal pathogens on grain yield of pre-released winter wheat genotypes in fungicide treated and untreated plots, and to determine whether S7b gene was present in ‘Gage’ wheat as previously hypothesized. Sixty winter wheat genotypes in fungicide treated and untreated plots were studied across four environments. There was a significant effect of fungicide on grain yield consistently across four environments in three years. Fungicide treated wheat lines demonstrated (4,496 kg/ ha-1) grain yield compared to (3,147 kg/ ha-1) grain yield in untreated wheat lines indicating 43% increased grain yield due to severity of foliar fungal diseases. Furthermore, fungicide application also caused an increase in protein concentration from 153 (g kg-1) to 164 (g kg-1) in treated plots in along with test weight from 73 to 77 (kg hL-1) respectively. Gage wheat variety and ISr7b-Ra were crossed to determine presence of Sr7b in Gage. The F2 and F2:3 segregating families were screened and evaluated for stem rust resistance. The segregation of families fell within 15:1 ratio for two separate resistance genes suggesting that Sr7b segregates independently from an unknown resistance gene in Gage that needs to be characterized for its use in the future wheat breeding program to develop resistant wheat varieties.

Keywords: funicide, genetics, foliar diseases, grain

Procedia PDF Downloads 126

Authors: I. N. Nwafia, U. C. Ozumba, M. E. Ohanu, S. O. Ebede

Abstract:

Antibiotic resistance is increasing globally and has become a major health challenge. Extended-spectrum beta-lactamase is clinically important because the ESBL gene are mostly plasmid encoded and these plasmids frequently carry genes encoding resistance to other classes of antimicrobials thereby limiting antibiotic options in the treatment of infections caused by these organisms. The specific objectives of this study were to determine the prevalence of ESBLs production in Escherichia coli, to determine the antibiotic susceptibility pattern of ESBLs producing Escherichia coli, to detect TEM, SHV and CTX-M genes and the risk factors to acquisition of ESBL producing Escherichia coli. The protocol of the study was approved by Health Research and Ethics committee of the University of Nigeria Teaching Hospital (UNTH), Enugu. It was a descriptive cross-sectional study that involved all hospitalized patients in UNTH from whose specimens Escherichia coli was isolated during the period of the study. The samples analysed were urine, wound swabs, blood and cerebrospinal fluid. These samples were cultured in 5% sheep Blood agar and MacConkey agar (Oxoid Laboratories, Cambridge UK) and incubated at 35-370C for 24 hours. Escherichia coli was identified with standard biochemical tests and confirmed using API 20E auxanogram (bioMerieux, Marcy 1'Etoile, France). The antibiotic susceptibility testing was done by disc diffusion method and interpreted according to the Clinical and Laboratory Standard Institute guideline. ESBL production was confirmed using ESBL Epsilometer test strips (Liofilchem srl, Italy). The ESBL bla genes were detected with polymerase chain reaction, after extraction of DNA with plasmid mini-prep kit (Jena Bioscience, Jena, Germany). Data analysis was with appropriate descriptive and inferential statistics. One hundred and six isolates (53.00%) out of the 200 were from urine, followed by isolates from different swabs specimens 53(26.50%) and the least number of the isolates 4(2.00) were from blood (P value = 0.096). Seventy (35.00%) out of the 200 isolates, were confirmed positive for ESBL production. Forty-two (60.00%) of the isolates were from female patients while 28(40.00%) were from male patients (P value = 0.13). Sixty-eight (97.14%) of the isolates were susceptible to imipenem while all of the isolates were resistant to ampicillin, chloramphenicol and tetracycline. From the 70 positive isolates the ESBL genes detected with polymerase chain reaction were blaCTX-M (n=26; 37.14%), blaTEM (n=7; 10.00%), blaSHV (n=2; 2.86%), blaCTX-M/TEM (n=7; 10.0%), blaCTX-M/SHV (n=14; 20.0%) and blaCTX-M/TEM/SHV (n=10; 14.29%). There was no gene detected in 4(5.71%) of the isolates. The most associated risk factors to infections caused by ESBL producing Escherichia coli was previous antibiotics use for the past 3 months followed by admission in the intensive care unit, recent surgery, and urinary catheterization. In conclusion, ESBLs was detected in 4 of every 10 Escherichia coli with the predominant gene detected being CTX-M. This knowledge will enable appropriate measures towards improvement of patient health care, antibiotic stewardship, research and infection control in the hospital.

Keywords: antimicrobial, Escherichia coli, extended spectrum beta lactamase, resistance

Procedia PDF Downloads 299

Authors: Neslihan Taskale Karatug, Mustafa Akcelik

Abstract:

Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

Procedia PDF Downloads 391

Authors: Sohan Sengupta, Arnab Pramanik, Abhrajyoti Ghosh, Maitree Bhattacharyya

Abstract:

Newly emerged phyto-pathogens and multi drug resistance have been threating the world for last few decades. Actinomycetes, the most endowed group of microorganisms isolated from unexplored regions of the world may be the ultimate solution to these problems. Thus the aim of this study was to isolate several bioactive actinomycetes strains capable of producing antimicrobial secondary metabolite from Sundarbans, the only mangrove tiger land of the world. Fifty four actinomycetes were isolated and analyzed for antimicrobial activity against fifteen test organisms including three phytopathogens. Nine morphologically distinct and biologically active isolates were subjected to polyphasic identification study. 16s rDNA sequencing indicated eight isolates to reveal maximum similarity to the genus streptomyces, whereas one isolate presented only 93.57% similarity with Streptomyces albogriseolus NRRL B-1305T. Seventy-one carbon sources and twenty-three chemical sources utilization assay revealed their metabolic relatedness. Among these nine isolates three specific strains were found to have notably higher degree of antimicrobial potential effective in a broader range including phyto-pathogenic fungus. PCR base whole genome screen for PKS and NRPS genes, confirmed the occurrence of bio-synthetic gene cluster in some of the isolates for novel antibiotic production. Finally the strain SMS_SU21, which showed antimicrobial activity with MIC value of 0.05 mg ml-1and antioxidant activity with IC50 value of 0.242±0.33 mg ml-1 was detected to be the most potential one. True prospective of this strain was evaluated utilizing GC-MS and the bioactive compound responsible for antimicrobial activity was purified and characterized. Rare bioactive actinomycetes were isolated from unexplored heritage site. Diversity of the biosynthetic gene cluster for antimicrobial compound production has also been evaluated. Antimicrobial compound SU21-C has been identified and purified which is active against a broad range of pathogens.

Keywords: actinomycetes, sundarbans, antimicrobial, pks nrps, phyto-pathogens, GC-MS

Procedia PDF Downloads 505

Authors: Nadia Naseem, Farwa Batool, Nasir Mehmood, AbdulHannan Nagi

Abstract:

Background: The incidence and mortality of breast cancer vary significantly in geographically distinct populations. In Pakistan, breast cancer has shown an increase in incidence in young females and is characterized by more aggressive behavior. The tumor suppressor TP53 gene is a crucial genetic factor that plays a significant role in breast carcinogenesis. This study investigated the TP53 mutations in molecular subtypes of both nodes negative and positive breast cancer in young Pakistani patients. Material and Methods: p53, Estrogen Receptor (ER), Progesterone Receptor (PR), Her-2 neu and Ki 67 expressions were analyzed immunohistochemically in a series of 75 node negative (A) and 75 node positive (B) young (aged: 19-40 years) breast cancer patients diagnosed between 2014 to 2017 at two leading hospitals of Punjab, Pakistan. Tumor tissue specimens and peripheral blood samples were examined for TP53 mutations by direct sequencing of the gene (exons 4-9). The relation of TP53 mutations to these markers and clinicopathological data was investigated. Results: Mean age of the patients was 32.4 + 9.1 SD. Invasive breast carcinoma was the most frequent histological variant (A=92%, B=94.6%). Grade 3 carcinoma was the commonest grade (A=72%, B=81.3%). Triple negative cases (ER-, PR-, Her-2) formed most of the molecular subtypes (A=44%, B=50.6%). A total of 17.2% (A: 6.6%, B: 10.6%) patients showed TP53 mutations. Mutations were significantly more frequent in triple negative cases (A: 74.8%, B: 62.2%) compared to HER2-positive patients (P < 0.0001). In the multivariate analysis of the whole patient group, the independent prognosticator were triple negative cases (P=0.021), TP53 overexpression by IHC (P=0.001) and advanced-stage disease (P=0.007). No statistically significant correlation between TP53 mutations and clinicopathological parameters was found (P < 0.05). Conclusions: It is concluded that TP53 mutations are infrequently present in breast carcinoma of young Pakistani population and there was no significant correlation between p53 mutation and early onset disease. Immunohistochemically detected TP53 expression in our resource-constrained to set up can be beneficial in predicting mutations at the younger age in our population.

Keywords: immunohistochemistry (IHC), invasive breast carcinoma (IBC), Pakistan, TP53

Procedia PDF Downloads 158

Authors: Hossein Rassi, Marjan Moradi Fard, Masoud Houshmand

Abstract:

Background: Cervical cancer is multistep disease that is thought to result from an interaction between genetic background and environmental factors. Human papillomavirus (HPV) infection is the leading risk factor for cervical intraepithelial neoplasia(CIN)and cervical cancer. In other hand, some of p53 and miRNA polymorphism may plays an important role in carcinogenesis. This study attempts to clarify the relation of p53 genotypes and miR-146a rs2910164 polymorphism in cervical lesions. Method: Forty two archival samples with cervical lesion retired from Khatam hospital and 40 sample from healthy persons used as control group. A simple and rapid method was used to detect the simultaneous amplification of the HPV consensus L1 region and HPV-16,-18, -11, -31, 33 and -35 along with the b-globin gene as an internal control. We use Multiplex PCR for detection of P53 and miR-146a rs2910164 genotypes in our lab. Finally, data analysis was performed using the 7 version of the Epi Info(TM) 2012 software and test chi-square(x2) for trend. Results: Cervix lesions were collected from 42 patients with Squamous metaplasia, cervical intraepithelial neoplasia, and cervical carcinoma. Successful DNA extraction was assessed by PCR amplification of b-actin gene (99bp). According to the results, p53 GG genotype and miR-146a rs2910164 CC genotype was significantly associated with increased risk of cervical lesions in the study population. In this study, we detected 13 HPV 18 from 42 cervical cancer. Conclusion: The connection between several SNP polymorphism and human virus papilloma in rare researches were seen. The reason of these differences in researches' findings can result in different kinds of races and geographic situations and also differences in life grooves in every region. The present study provided preliminary evidence that a p53 GG genotype and miR-146a rs2910164 CC genotype may effect cervical cancer risk in the study population, interacting synergistically with HPV 18 genotype. Our results demonstrate that the testing of p53 codon 72 polymorphism genotypes and miR-146a rs2910164 polymorphism genotypes in combination with HPV18 can serve as major risk factors in the early identification of cervical cancers. Furthermore, the results indicate the possibility of primary prevention of cervical cancer by vaccination against HPV18 in Iran.

Keywords: cervical cancer, p53, miR-146a, rs2910164, polymorphism

Procedia PDF Downloads 468

Authors: Arsyam Mawardi, Sony Suhandono, Azzania Fibriani, Fifi Fitriyah Masduki

Abstract:

Malaria is an infectious disease caused by Plasmodium sp. This disease has a high prevalence in Indonesia, especially in Jayapura. The vaccine that is currently being developed has not been effective in overcoming malaria. This is due to the high polymorphism in the Plasmodium genome especially in areas that encode Plasmodium surface proteins. Merozoite Surface Protein 1 (MSP1) Plasmodium falciparum is a surface protein that plays a role in the invasion process in human erythrocytes through the interaction of Glycophorin A protein receptors and sialic acid in erythrocytes with Reticulocyte Binding Proteins (RBP) and Duffy Adhesion Protein (DAP) ligands in merozoites. MSP1 can be targeted to be a specific antigen and predicted epitope area which will be used for the development of diagnostic and malaria vaccine therapy. MSP1 consists of 17 blocks, each block is dimorphic, and has been marked as the K1 and MAD20 alleles. Exceptions only in block 2, because it has 3 alleles, among others K1, MAD20 and RO33. These polymorphisms cause allelic variations and implicate the severity of patients infected P. falciparum. In addition, polymorphism of MSP1 in Jayapura isolates has not been reported so it is interesting to be further identified and projected as a specific antigen. Therefore, in this study, we analyzed the allele polymorphism as well as detected the MSP1 epitope antigen candidate on block 2 P. falciparum. Clinical samples of selected malaria patients followed the consecutive sampling method, examining malaria parasites with blood preparations on glass objects observed through a microscope. Plasmodium DNA was isolated from the blood of malarial positive patients. The block 2 MSP1 gene was amplified using PCR method and cloned using the pGEM-T easy vector then transformed to TOP'10 E.coli. Positive colonies selection was performed with blue-white screening. The existence of target DNA was confirmed by PCR colonies and DNA sequencing methods. Furthermore, DNA sequence analysis was done through alignment and formation of a phylogenetic tree using MEGA 6 software and insilico analysis using IEDB software to predict epitope candidate for P. falciparum. A total of 15 patient samples have been isolated from Plasmodium DNA. PCR amplification results show the target gene size about ± 1049 bp. The results of MSP1 nucleotide alignment analysis reveal that block 2 MSP1 genes derived from the sample of malarial patients were distributed in four different allele family groups, K1 (7), MAD20 (1), RO33 (0) and MSP1_Jayapura (10) alleles. The most commonly appears of the detected allele is MSP1_Jayapura single allele. There was no significant association between sex variables, age, the density of parasitemia and alel variation (Mann Whitney, U > 0.05), while symptomatic signs have a significant difference as a trigger of detectable allele variation (U < 0.05). In this research, insilico study shows that there is a new epitope antigen candidate from the MSP1_Jayapura allele and it is predicted to be recognized by B cells with 17 amino acid lengths in the amino acid sequence 187 to 203.

Keywords: epitope candidate, insilico analysis, MSP1 P. falciparum, polymorphism

Procedia PDF Downloads 180

Authors: Hossein Rassi, Marjan Moradi fard, Masoud Houshmand

Abstract:

Background: Cervical cancer is multistep disease that is thought to result from an interaction between genetic background and environmental factors. Human Papillomavirus (HPV) infection is the leading risk factor for Cervical Intraepithelial Neoplasia (CIN) and cervical cancer. In other hand, some of p53 and miRNA polymorphism may plays an important role in carcinogenesis. This study attempts to clarify the relation of p53 genotypes and miR-146a rs2910164 polymorphism in cervical lesions. Method: Forty two archival samples with cervical lesion retired from Khatam hospital and 40 sample from healthy persons used as control group. A simple and rapid method was used to detect the simultaneous amplification of the HPV consensus L1 region and HPV-16,-18, -11, -31, 33, and -35 along with the b-globin gene as an internal control. We use Multiplex PCR for detection of P53 and miR-146a rs2910164 genotypes in our lab. Finally, data analysis was performed using the 7 version of the Epi Info(TM) 2012 software and test chi-square(x2) for trend. Results: Cervix lesions were collected from 42 patients with Squamous metaplasia, cervical intraepithelial neoplasia, and cervical carcinoma. Successful DNA extraction was assessed by PCR amplification of b-actin gene (99 bp). According to the results, p53 GG genotype and miR-146a rs2910164 CC genotype was significantly associated with increased risk of cervical lesions in the study population. In this study, we detected 13 HPV 18 from 42 cervical cancer. Conclusion: The connection between several SNP polymorphism and human virus papilloma in rare researches were seen. The reason of these differences in researches' findings can result in different kinds of races and geographic situations and also differences in life grooves in every region. The present study provided preliminary evidence that a p53 GG genotype and miR-146a rs2910164 CC genotype may effect cervical cancer risk in the study population, interacting synergistically with HPV 18 genotype. Our results demonstrate that the testing of p53 codon 72 polymorphism genotypes and miR-146a rs2910164 polymorphism genotypes in combination with HPV18 can serve as major risk factors in the early identification of cervical cancers. Furthermore, the results indicate the possibility of primary prevention of cervical cancer by vaccination against HPV18 in Iran.

Keywords: cervical cancer, miR-146a rs2910164 polymorphism, p53 polymorphism, intraepithelial, neoplasia, HPV

Procedia PDF Downloads 398

Authors: German Hernandez-Alonso, Antonio Lazcano, Arturo Becerra

Abstract:

Until today, viruses remain controversial and poorly understood; about their origin, this problem represents an enigma and one of the great challenges for the contemporary biology. Three main theories have tried to explain the origin of viruses: regressive evolution, escaped host gene, and pre-cellular origin. Under the perspective of the escaped host gene theory, it can be assumed a cellular origin of viral components, like protein RNA-binding domains. These universal distributed RNA-binding domains are related to the RNA metabolism processes, including transcription, processing, and modification of transcripts, translation, RNA degradation and its regulation. In the case of viruses, these domains are present in important viral proteins like helicases, nucleases, polymerases, capsid proteins or regulation factors. Therefore, they are implicated in the replicative cycle and parasitic processes of viruses. That is why it is possible to think that those domains present low levels of divergence due to selective pressures. For these reasons, the main goal for this project is to create a catalogue of the RNA-binding domains found in all the available viral proteomes, using bioinformatics tools in order to analyze its evolutionary process, and thus shed light on the general virus evolution. ProDom database was used to obtain larger than six thousand RNA-binding domain families that belong to the three cellular domains of life and some viral groups. From the sequences of these families, protein profiles were created using HMMER 3.1 tools in order to find distant homologous within greater than four thousand viral proteomes available in GenBank. Once accomplished the analysis, almost three thousand hits were obtained in the viral proteomes. The homologous sequences were found in proteomes of the principal Baltimore viral groups, showing interesting distribution patterns that can contribute to understand the evolution of viruses and their host-virus interactions. Presence of cellular RNA-binding domains within virus proteomes seem to be explained by closed interactions between viruses and their hosts. Recruitment of these domains is advantageous for the viral fitness, allowing viruses to be adapted to the host cellular environment.

Keywords: bioinformatics tools, distant homology, RNA-binding domains, viral evolution

Procedia PDF Downloads 387

Authors: H. Haseena Banu, D. Karthick, R. Stalin, E. Nandha Kumar, T. P. Sachidanandam, P. Shanthi

Abstract:

Background of the study: Type 2 diabetes is emerging as the predominant metabolic disorder in the world among adults characterized mainly by the resistance of the insulin sensitive tissues towards insulin followed by the decrease in the insulin secretion. The treatment for this disease usually involves treatment with oral synthetic drugs which are known to cause several side effects. Therefore, identification of new biomarkers as therapeutic target is the need of the hour. miRNAs are small, non–protein-coding RNAs that negatively regulate gene expression by promoting degradation and/or inhibit the translation of target mRNAs and have emerged as biomarkers in predicting diabetes mellitus. Objective of the study: To elucidate the therapeutic role of gallic acid in modulating the alterations in glucose metabolism induced by miRNAs 194 and 135a in Type 2 diabetic rats. Materials and Methods: T2D was induced in rats by feeding them with a high fat diet for 2 weeks followed by intraperitoneal injection of 35 mg/kg/body weight (b.wt.) of streptozotocin. Microarrays were used to assess the expression of miRNAs in control, diabetic and gallic acid treated rats. Gene expression studies were carried out by RT PCR analysis. Results: Forty one miRNAs were differentially expressed in Type 2 diabetic rats. Among these, the expression of miRNA 194 was significantly decreased whereas miRNA 135a was significantly increased in Type 2 diabetic rats. The glucose metabolism was also altered significantly in skeletal muscle of Type 2 diabetic rats. Conclusion: T2D is associated with alterations in the expression of miRNAs in skeletal muscle. Both these miRNAs 194 and 135a play an important role in glucose metabolism in skeletal muscle of diabetic rats. Gallic acid effectively ameliorated the alterations in glucose metabolism. Hence, both these miRNAs can serve as biomarkers and therapeutic targets in diabetes mellitus. The study also establishes the role of gallic acid as therapeutic agent. Acknowledgment: The financial assistance provided in the form of ICMR women scientist by ICMR DHR INDIA is gratefully acknowledged here.

Keywords: gallic acid, high fat diet, type 2 diabetes mellitus, miRNAs

Procedia PDF Downloads 349

Authors: Salman Akrokayan

Abstract:

Synthetic cDNA (ScDNA) of granulocyte colony-stimulating factor (G-CSF) was constructed using a DNA synthesizer with the aim to increase its expression level. 5' end of the ScDNA of G-CSF coding region was modified by decreasing the GC content without altering the predicted amino acids sequence. The identity of the resulting protein from ScDNA was confirmed by the highly specific enzyme-linked immunosorbent assay. In conclusion, a synthetic G-CSF cDNA in combination with the recombinant DNA protocol offers a rapid and reliable strategy for synthesizing the target protein. However, the commercial utilization of this methodology requires rigorous validation and quality control.

Keywords: synthetic cDNA, recombinant G-CSF, cloning, gene expression

Procedia PDF Downloads 284