Search results for: gene profiling

Authors: Ankita Singh, Chandan Gorain, Amirul I. Mallick

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Successful adherence of the mucosal epithelial cells is the key early step for Campylobacter jejuni pathogenesis (C. jejuni). A set of Surface Exposed Colonization Proteins (SECPs) are among the major factors involved in host cell adherence and invasion of C. jejuni. Among them, constitutively expressed surface-exposed lipoprotein adhesin of C. jejuni, JlpA, interacts with intestinal heat shock protein 90 (hsp90α) and contributes in disease progression by triggering pro-inflammatory response via activation of NF-κB and p38 MAP kinase pathway. Together with its ability to express in the bacterial surface, higher sequence conservation and predicted predominance of several B cells epitopes, JlpA protein reserves its potential to become an effective vaccine candidate against wide range of Campylobacter sps including C. jejuni. Given that chickens are the primary sources for C. jejuni and persistent gut colonization remain as major cause for foodborne pathogenesis to humans, present study explicitly used chickens as model to test the immune-protective efficacy of JlpA protein. Taking into account that gastrointestinal tract is the focal site for C. jejuni colonization, to extrapolate the benefit of mucosal (intragastric) delivery of JlpA protein, a food grade Nisin inducible Lactic acid producing bacteria, Lactococcus lactis (L. lactis) was engineered to express recombinant JlpA protein (rJlpA) in the surface of the bacteria. Following evaluation of optimal surface expression and functionality of recombinant JlpA protein expressed by recombinant L. lactis (rL. lactis), the immune-protective role of intragastric administration of live rL. lactis was assessed in commercial broiler chickens. In addition to the significant elevation of antigen specific mucosal immune responses in the intestine of chickens that received three doses of rL. lactis, marked upregulation of Toll-like receptor 2 (TLR2) gene expression in association with mixed pro-inflammatory responses (both Th1 and Th17 type) was observed. Furthermore, intragastric delivery of rJlpA expressed by rL. lactis, but not the injectable form, resulted in a significant reduction in C. jejuni colonization in chickens suggesting that mucosal delivery of live rL. lactis expressing JlpA serves as a promising vaccine platform to induce strong immune-protective responses against C. jejuni in chickens.

Keywords: chickens, lipoprotein adhesion of Campylobacter jejuni, immuno-protection, Lactococcus lactis, mucosal delivery

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Authors: Kerekoppa Puttaiah Bhatta Ramesha, Uday Kannegundla, Praseeda Mol, Lathika Gopalakrishnan, Jagish Kour Reen, Gourav Dey, Manish Kumar, Sakthivel Jeyakumar, Arumugam Kumaresan, Kiran Kumar M., Thottethodi Subrahmanya Keshava Prasad

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Spermatozoa are the highly specialized transcriptionally and translationally inactive haploid male gamete. The understanding of proteome of sperm is indispensable to explore the mechanism of sperm motility and fertility. Though there is a large number of human sperm proteomic studies, in-depth proteomic information on Bos indicus spermatozoa is not well established yet. Therefore, we illustrated the profile of sperm proteome in indigenous cattle, Malnad gidda (Bos Indicus), using high-resolution mass spectrometry. In the current study, two semen ejaculates from 3 breeding bulls were collected employing the artificial vaginal method. Using 45% percoll purification, spermatozoa cells were isolated. Protein was extracted using lysis buffer containing 2% Sodium Dodecyl Sulphate (SDS) and protein concentration was estimated. Fifty micrograms of protein from each individual were pooled for further downstream processing. Pooled sample was fractionated using SDS-Poly Acrylamide Gel Electrophoresis, which is followed by in-gel digestion. The peptides were subjected to C18 Stage Tip clean-up and analyzed in Orbitrap Fusion Tribrid mass spectrometer interfaced with Proxeon Easy-nano LC II system (Thermo Scientific, Bremen, Germany). We identified a total of 6773 peptides with 28426 peptide spectral matches, which belonged to 1081 proteins. Gene ontology analysis has been carried out to determine the biological processes, molecular functions and cellular components associated with sperm protein. The biological process chiefly represented our data is an oxidation-reduction process (5%), spermatogenesis (2.5%) and spermatid development (1.4%). The highlighted molecular functions are ATP, and GTP binding (14%) and the prominent cellular components most observed in our data were nuclear membrane (1.5%), acrosomal vesicle (1.4%), and motile cilium (1.3%). Seventeen percent of sperm proteins identified in this study were involved in metabolic pathways. To the best of our knowledge, this data represents the first total sperm proteome from indigenous cattle, Malnad Gidda. We believe that our preliminary findings could provide a strong base for the future understanding of bovine sperm proteomics.

Keywords: Bos indicus, Malnad Gidda, mass spectrometry, spermatozoa

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Authors: Indu Gaur, Neha Bhadauria, Shilpi Shilpi, Susmita Goswami, Prem D. Sharma, Prabir K. Paul

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The concept of organic agriculture has been accepted as novelty in Indian society, but there is no data available on the human pathogens colonizing plant parts due to such practices. Also, the pattern and mechanism of their colonization need to be understood in order to devise possible strategies for their prevention. In the present study, human pathogenic bacteria were isolated from organically grown tomato plants and five of them were identified as Klebsiella pneumoniae, Enterobacter ludwigii, Serratia fonticola, Stenotrophomonas maltophilia and Chryseobacterium jejuense. Tomato plants were grown in controlled aseptic conditions with 25±1˚C, 70% humidity and 12 hour L/D photoperiod. Six weeks old plants were divided into 6 groups of 25 plants each and treated as follows: Group 1: K. pneumonia, Group 2: E. ludwigii, Group 3: S. fonticola, Group 4: S. maltophilia, Group 5: C. jejuense, Group 6: Sterile distilled water (control). The inoculums for all treatments were prepared by overnight growth with uniform concentration of 108 cells/ml. Leaf samples from above groups were collected at 0.5, 2, 4, 6 and 24 hours post inoculation for the colony forming unit counts (CFU/cm2 of leaf area) of individual pathogens using leaf impression method. These CFU counts were used for the in vivo colonization assay and adherence assay of individual pathogens. Also, resistance of these pathogens to at least 12 antibiotics was studied. Based on these findings S. fonticola was found to be most prominently colonizing the phylloplane of tomato and was further studied. Tomato plants grown in controlled aseptic conditions same as mentioned above were divided into 2 groups of 25 plants each and treated as follows: Group 1: S. fonticola, Group 2: Sterile distilled water (control). Leaf samples from above groups were collected at 0, 24, 48, 72 and 96 hours post inoculation and homogenized in suitable buffers for surface and cell wall protein isolation. Protein samples thus obtained were subjected to isocratic SDS-gel electrophoresis and analyzed. It was observed that presence of S. fonticola could induce the expression of at least 3 additional cell wall proteins at different time intervals. Surface proteins also showed variation in the expression pattern at different sampling intervals. Further identification of these proteins by MALDI-MS and bioinformatics tools revealed the gene(s) involved in the interaction of S. fonticola with tomato phylloplane.

Keywords: cell wall proteins, human pathogenic bacteria, phylloplane, solanum lycopersicum

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Authors: Alan Ruiz-Padilla, Brando Villalobos-Villalobos, Yeniley Ruiz-Noa, Claudia Mendoza-Macías, Claudia Palafox-Sánchez, Miguel Marín-Rosales, Álvaro Cruz, Rubén Rangel-Salazar

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Introduction: In patients with rheumatoid arthritis, resistance or poor response to disease modifying antirheumatic drugs (DMARD) may be a reflection of the increase in g-P. The expression of g-P may be important in mediating the effluence of DMARD from the cell. In addition, P-glycoprotein is involved in the transport of cytokines, IL-1, IL-2 and IL-4, from normal lymphocytes activated to the surrounding extracellular matrix, thus influencing the activity of RA. The involvement of P-glycoprotein in the transmembrane transport of cytokines can serve as a modulator of the efficacy of DMARD. It was shown that a number of lymphocytes with glycoprotein P activity is increased in patients with RA; therefore, P-glycoprotein expression could be related to the activity of RA and could be a predictor of poor response to therapy. Objective: To evaluate in RA patients, if the G2677T/A MDR1 polymorphisms is associated with differences in the rate of therapeutic response to disease-modifying antirheumatic agents in patients with rheumatoid arthritis. Material and Methods: A prospective cohort study was conducted. Fifty seven patients with RA were included. They had an active disease according to DAS-28 (score >3.2). We excluded patients receiving biological agents. All the patients were followed during 6 months in order to identify the rate of therapeutic response according to the American College of Rheumatology (ACR) criteria. At the baseline peripheral blood samples were taken in order to identify the G2677T/A MDR1 polymorphisms using PCR- Specific allele. The fragment was identified by electrophoresis in polyacrylamide gels stained with ethidium bromide. For statistical analysis, the genotypic and allelic frequencies of MDR1 gene polymorphism between responders and non-responders were determined. Chi-square tests as well as, relative risks with 95% confidence intervals (95%CI) were computed to identify differences in the risk for achieving therapeutic response. Results: RA patients had a mean age of 47.33 ± 12.52 years, 87.7% were women with a mean for DAS-28 score of 6.45 ± 1.12. At the 6 months, the rate of therapeutic response was 68.7 %. The observed genotype frequencies were: for G/G 40%, T/T 32%, A/A 19%, G/T 7% and for A/A genotype 2%. Patients with G allele developed at 6 months of treatment, higher rate for therapeutic response assessed by ACR20 compared to patients with others alleles (p=0.039). Conclusions: Patients with G allele of the - G2677T/A MDR1 polymorphisms had a higher rate of therapeutic response at 6 months with DMARD. These preliminary data support the requirement for a deep evaluation of these and other genotypes as factors that may influence the therapeutic response in RA.

Keywords: pharmacogenetics, MDR1, P-glycoprotein, therapeutic response, rheumatoid arthritis

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Authors: Suchhanda Ghosh, A. K. Paul

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Nickel, an industrially important metal is not mined in India, due to the lack of its primary mining resources. But, the chromite deposits occurring in the Sukinda and Baula-Nuasahi region of Odhisa, India, is reported to contain around 0.99% of nickel entrapped in the goethite matrix of the lateritic iron rich ore. Weathering of the dumped chromite mining overburden often leads to the contamination of the ground as well as the surface water with toxic nickel. Microbes inherent to this metal contaminated environment are reported to be capable of removal as well as detoxification of various metals including nickel. Nickel resistant fungal isolates obtained in pure form from the metal rich overburden were evaluated for their potential to biosorb nickel by using their dried biomass. Penicillium simplicissimum SAU203 was the best nickel biosorbant among the 20 fungi tested and was capable to sorbing 16.85 mg Ni/g biomass from a solution containing 50 mg/l of Ni. The identity of the isolate was confirmed using 18S rRNA gene analysis. The sorption capacity of the isolate was further standardized following Langmuir and Freundlich adsorption isotherm models and the results reflected energy efficient sorption. Fourier-transform infrared spectroscopy studies of the nickel loaded and control biomass in a comparative basis revealed the involvement of hydroxyl, amine and carboxylic groups in Ni binding. The sorption process was also optimized for several standard parameters like initial metal ion concentration, initial sorbet concentration, incubation temperature and pH, presence of additional cations and pre-treatment of the biomass by different chemicals. Optimisation leads to significant improvements in the process of nickel biosorption on to the fungal biomass. P. simplicissimum SAU203 could sorb 54.73 mg Ni/g biomass with an initial Ni concentration of 200 mg/l in solution and 21.8 mg Ni/g biomass with an initial biomass concentration of 1g/l solution. Optimum temperature and pH for biosorption was recorded to be 30°C and pH 6.5 respectively. Presence of Zn and Fe ions improved the sorption of Ni(II), whereas, cobalt had a negative impact. Pre-treatment of biomass with various chemical and physical agents has affected the proficiency of Ni sorption by P. simplicissimum SAU203 biomass, autoclaving as well as treatment of biomass with 0.5 M sulfuric acid and acetic acid reduced the sorption as compared to the untreated biomass, whereas, NaOH and Na₂CO₃ and Twin 80 (0.5 M) treated biomass resulted in augmented metal sorption. Hence, on the basis of the present study, it can be concluded that P. simplicissimum SAU203 has the potential for the removal as well as detoxification of nickel from contaminated environments in general and particularly from the chromite mining areas of Odhisa, India.

Keywords: nickel, fungal biosorption, Penicillium simplicissimum SAU203, Indian chromite mines, mining overburden

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Authors: Shraddha Rane, Richard Warren, Stephen Eyre

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L-23 is a pro-inflammatory molecule that signals T cells to release cytokines such as IL-17A and IL-22. Psoriasis is driven by a dysregulated immune response, within which IL-23 is now thought to play a key role. Genome-wide association studies (GWAS) have identified a number of genetic risk loci that support the involvement of IL-23 signalling in psoriasis; in particular a robust susceptibility locus at a gene encoding a subunit of the IL-23 receptor (IL23R) (Stuart et al., 2015; Tsoi et al., 2012). The lead psoriasis-associated SNP rs9988642 is located approximately 500 bp downstream of IL23R but is in tight linkage disequilibrium (LD) with a missense SNP rs11209026 (R381Q) within IL23R (r2 = 0.85). The minor (G) allele of rs11209026 is present in approximately 7% of the population and is protective for psoriasis and several other autoimmune diseases including IBD, ankylosing spondylitis, RA and asthma. The psoriasis-associated missense SNP R381Q causes an arginine to glutamine substitution in a region of the IL23R protein between the transmembrane domain and the putative JAK2 binding site in the cytoplasmic portion. This substitution is expected to affect the receptor’s surface localisation or signalling ability, rather than IL23R expression. Recent studies have also identified a psoriatic arthritis (PsA)-specific signal at IL23R; thought to be independent from the psoriasis association (Bowes et al., 2015; Budu-Aggrey et al., 2016). The lead PsA-associated SNP rs12044149 is intronic to IL23R and is in LD with likely causal SNPs intersecting promoter and enhancer marks in memory CD8+ T cells (Budu-Aggrey et al., 2016). It is therefore likely that the PsA-specific SNPs affect IL23R function via a different mechanism compared with the psoriasis-specific SNPs. It could be hypothesised that the risk allele for PsA located within the IL23R promoter causes an increase IL23R expression, relative to the protective allele. An increased expression of IL23R might then lead to an exaggerated immune response. The independent genetic signals identified for psoriasis and PsA in this locus indicate that different mechanisms underlie these two conditions; although likely both affecting the function of IL23R. It is very important to further characterise these mechanisms in order to better understand how the IL-23 receptor and its downstream signalling is affected in both diseases. This will help to determine how psoriasis and PsA patients might differentially respond to therapies, particularly IL-23 biologics. To investigate this further we have developed an in vitro model using CD4 T cells which express either wild type IL23R and IL12Rβ1 or mutant IL23R (R381Q) and IL12Rβ1. Model expressing different isotypes of IL23R is also underway to investigate the effects on IL23R expression. We propose to further investigate the variants for Ps and PsA and characterise key intracellular processes related to the variants.

Keywords: IL23R, psoriasis, psoriatic arthritis, SNP

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Authors: R. Pussinen, V. Jankovic, U. Herzberg, M. Cerrada-Gimenez, T. Huhtala, A. Nurmi, T. Ahtoniemi

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Targeted over-expression of human α-synuclein using viral-vector mediated gene delivery into the substantia nigra of rats and non-human primates has been reported to lead to dopaminergic cell loss and the formation of α-synuclein aggregates reminiscent of Lewy bodies. We have previously shown how AAV-mediated expression of α-synuclein is seen in the chronic phenotype of the rats over 16 week follow-up period. In the context of these findings, we attempted to further characterize this long term PD related functional and motor deficits as well as neurochemical and neuropathological changes in AAV-mediated α-synuclein transfection model in rats during chronic follow-up period. Different titers of recombinant AAV expressing human α-synuclein (A53T) were stereotaxically injected unilaterally into substantia nigra of Wistar rats. Rats were allowed to recover for 3 weeks prior to initial baseline behavioral testing with rotational asymmetry test, stepping test and cylinder test. A similar behavioral test battery was applied again at weeks 5, 9,12 and 15. In addition to traditionally used rat PD model tests, MotoRater test system, a high speed kinematic gait performance monitoring was applied during the follow-up period. Evaluation focused on animal gait between groups. Tremor analysis was performed on weeks 9, 12 and 15. In addition to behavioral end-points, neurochemical evaluation of dopamine and its metabolites were evaluated in striatum. Furthermore, integrity of the dopamine active transport (DAT) system was evaluated by using 123I- β-CIT and SPECT/CT imaging on weeks 3, 8 and 12 after AAV- α-synuclein transfection. Histopathology was examined from end-point samples at 3 or 12 weeks after AAV- α-synuclein transfection to evaluate dopaminergic cell viability and microglial (Iba-1) activation status in substantia nigra by using stereological analysis techniques. This study focused on the characterization and validation of previously published AAV- α-synuclein transfection model in rats but with the addition of novel end-points. We present the long term phenotype of AAV- α-synuclein transfected rats with traditionally used behavioral tests but also by using novel fine motor analysis techniques and tremor analysis which provide new insight to unilateral effects of AAV α-synuclein transfection. We also present data about neurochemical and neuropathological end-points for the dopaminergic system in the model and how well they correlate with behavioral phenotype.

Keywords: adeno-associated virus, alphasynuclein, animal model, Parkinson’s disease

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

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For chemotherapy, a drug delivery system should be able to specifically target cancer cells and deliver the therapeutic dose without affecting normal cells. Folate receptors (FR) can be considered key targets since they are commonly over-expressed in cancer cells and they are the molecular marker used in this study. Here, cyclodextrin (CD) has being studied as a vehicle for delivering the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within the CD cavity. In this study, β-CD has been modified using folic acid so as to specifically target the FR molecular marker. Thus, the system studied here for drug delivery consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 9.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 10.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 8.5 for A549 and 132.6 µM ± 12.1 and 288.1 µM ± 16.3 for BEAS-2B. These results demonstrate that MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug.

Keywords: cyclodextrins, cancer treatment, drug delivery, folate receptors, reduced folate carriers

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Authors: Xi Chen, King-Yip Cheng

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Hypercholesterolemia, characterized by high low-density lipoprotein cholesterol (LDL-C), raises cardiovascular events in patients with type 2 diabetes (T2D). Although several drugs, such as statin and PCSK9 inhibitors, are available for the treatment of hypercholesterolemia, they exert detrimental effects on glucose metabolism and hence increase the risk of T2D. On the other hand, the drugs used to treat T2D have minimal effect on improving the lipid profile. Therefore, there is an urgent need to develop treatments that can simultaneously improve glucose and lipid homeostasis. Adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 2 (APPL2) causes insulin resistance in the liver and skeletal muscle via inhibiting insulin and adiponectin actions in animal models. Single-nucleotide polymorphisms in the APPL2 gene were associated with LDL-C, non-alcoholic fatty liver disease, and coronary artery disease in humans. The aim of this project is to investigate whether APPL2 antisense oligonucleotide (ASO) can alleviate dietary-induced T2D and hypercholesterolemia. High-fat diet (HFD) was used to induce obesity and insulin resistance in mice. GalNAc-conjugated APPL2 ASO (GalNAc-APPL2-ASO) was used to silence hepatic APPL2 expression in C57/BL6J mice selectively. Glucose, lipid, and energy metabolism were monitored. Immunoblotting and quantitative PCR analysis showed that GalNAc-APPL2-ASO treatment selectively reduced APPL2 expression in the liver instead of other tissues, like adipose tissues, kidneys, muscle, and heart. The glucose tolerance test and insulin sensitivity test revealed that GalNAc-APPL2-ASO improved glucose tolerance and insulin sensitivity progressively. Blood chemistry analysis revealed that the mice treated with GalNAc-APPL2-ASO had significantly lower circulating levels of total cholesterol and LDL cholesterol. However, there was no difference in circulating levels of high-density lipoprotein (HDL) cholesterol, triglyceride, and free fatty acid between the mice treated with GalNac-APPL2-ASO and GalNAc-Control-ASO. No obvious effect on food intake, body weight, and liver injury markers after GalNAc-APPL2-ASO treatment was found, supporting its tolerability and safety. We showed that selectively silencing hepatic APPL2 alleviated insulin resistance and hypercholesterolemia and improved energy metabolism in the dietary-induced obese mouse model, indicating APPL2 as a therapeutic target for metabolic diseases.

Keywords: APPL2, antisense oligonucleotide, hypercholesterolemia, type 2 diabetes

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Authors: Irina M. Kolesnikova, Andrey M. Gaponov, Sergey A. Roumiantsev, Tatiana V. Grigoryeva, Dilyara R. Khusnutdinova, Dilyara R. Kamaldinova, Alexander V. Shestopalov

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Background. Obese patients have unequal risks of metabolic disorders. It is accepted to distinguish between metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUHO). MUHO patients have a high risk of metabolic disorders, insulin resistance, and diabetes mellitus. Among the other things, the gut microbiota also contributes to the development of metabolic disorders in obesity. Obesity is accompanied by significant changes in the gut microbial community. In turn, bacterial translocation from the intestine is the basis for the blood microbiome formation. The aim was to study the features of the blood microbiome in patients with various metabolic types of obesity. Patients, materials, methods. The study included 116 healthy donors and 101 obese patients. Depending on the metabolic type of obesity, the obese patients were divided into subgroups with MHO (n=36) and MUHO (n=53). Quantitative and qualitative assessment of the blood microbiome was based on metagenomic analysis. Blood samples were used to isolate DNA and perform sequencing of the variable v3-v4 region of the 16S rRNA gene. Alpha diversity indices (Simpson index, Shannon index, Chao1 index, phylogenetic diversity, the number of observed operational taxonomic units) were calculated. Moreover, we compared taxa (phyla, classes, orders, and families) in terms of isolation frequency and the taxon share in the total bacterial DNA pool between different patient groups. Results. In patients with MHO, the characteristics of the alpha-diversity of the blood microbiome were like those of healthy donors. However, MUHO was associated with an increase in all diversity indices. The main phyla of the blood microbiome were Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Cyanobacteria, TM7, Thermi, Verrucomicrobia, Chloroflexi, Acidobacteria, Planctomycetes, Gemmatimonadetes, and Tenericutes were found to be less significant phyla of the blood microbiome. Phyla Acidobacteria, TM7, and Verrucomicrobia were more often isolated in blood samples of patients with MUHO compared with healthy donors. Obese patients had a decrease in some taxonomic ranks (Bacilli, Caulobacteraceae, Barnesiellaceae, Rikenellaceae, Williamsiaceae). These changes appear to be related to the increased diversity of the blood microbiome observed in obesity. An increase of Lachnospiraceae, Succinivibrionaceae, Prevotellaceae, and S24-7 was noted for MUHO patients, which, apparently, is explained by a magnification in intestinal permeability. Conclusion. Blood microbiome differs in obese patients and healthy donors at class, order, and family levels. Moreover, the nature of the changes is determined by the metabolic type of obesity. MUHO linked to increased diversity of the blood microbiome. This appears to be due to increased microbial translocation from the intestine and non-intestinal sources.

Keywords: blood microbiome, blood bacterial DNA, obesity, metabolically healthy obesity, metabolically unhealthy obesity

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Authors: Priyanka Bhowmik, Subrata Majumdar, Debprasad Chattopadhyay

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Background: Cervical cancer is the third major cause of cancer in women and the second most frequent cause of cancer related deaths causing 300,000 deaths annually worldwide. Evasion of immune response by Human Papilloma Virus (HPV), the key contributing factor behind cancer and pre-cancerous lesions of the uterine cervix, makes immunotherapy a necessity to treat this disease. Objective: A Heat killed fraction of Mycobacterium indicus pranii (MIP), a non-pathogenic Mycobacterium has been shown to exhibit cytotoxic effects on different cancer cells, including human cervical carcinoma cell line HeLa. However, the underlying mechanisms remain unknown. The aim of this study is to decipher the mechanism of MIP induced HeLa cell death. Methods: The cytotoxicity of Mycobacterium indicus pranii against HeLa cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and Propidium iodide (PI) staining. The assessment of reactive oxygen species (ROS) generation and cell cycle analysis were measured by flow cytometry. The expression of apoptosis associated genes was analyzed by real time PCR. Result: MIP could inhibit the proliferation of HeLa cell in a time and dose dependent manner but caused minor damage to normal cells. The induction of apoptosis was confirmed by the cell surface presentation of phosphatidyl serine, DNA fragmentation, and mitochondrial damage. MIP caused very early (as early as 30 minutes) transcriptional activation of p53, followed by a higher activation (32 fold) at 24 hours suggesting prime importance of p53 in MIP-induced apoptosis in HeLa cell. The up regulation of p53 dependent pro-apoptotic genes Bax, Bak, PUMA, and Noxa followed a lag phase that was required for the transcriptional p53 program. MIP also caused the transcriptional up regulation of Toll like receptor 2 and 4 after 30 minutes of MIP treatment suggesting recognition of MIP by toll like receptors. Moreover, MIP caused the inhibition of expression of HPV anti apoptotic gene E6, which is known to interfere with p53/PUMA/Bax apoptotic cascade. This inhibition might have played a role in transcriptional up regulation of PUMA and subsequently apoptosis. ROS was generated transiently which was concomitant with the highest transcription activation of p53 suggesting a plausible feedback loop network of p53 and ROS in the apoptosis of HeLa cells. Scavenger of ROS, such as N-acetyl-L-cysteine, decreased apoptosis suggesting ROS is an important effector of MIP induced apoptosis. Conclusion: Taken together, MIP possesses full potential to be a novel therapeutic agent in the clinical treatment of cervical cancer.

Keywords: cancer, mycobacterium, immunity, immunotherapy.

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Authors: B. Gonzalez-Yebra, H. Barajas, P. Palomares, M. Hernandez, O. Torres, M. Ayala, A. L. González, G. Vazquez-Ortiz, M. L. Guzman

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Leukemia arises by molecular alterations of the normal hematopoietic stem cell (HSC) transforming it into a leukemic stem cell (LSC) with high cell proliferation, self-renewal, and cell differentiation. Chronic myeloid leukemia (CML) originates from an LSC-leading to elevated proliferation of myeloid cells and acute lymphoblastic leukemia (ALL) originates from an LSC development leading to elevated proliferation of lymphoid cells. In both cases, LSC can be identified by multicolor flow cytometry using several antibodies. However, to date, LSC levels in peripheral blood (PB) are not established well enough in ALL and CML patients. On the other hand, the detection of the minimal residue disease (MRD) in leukemia is mainly based on the identification of the mRNA bcr-abl gene in CML patients and some other genes in ALL patients. There is no a properly biomarker to detect MDR in both types of leukemia. The objective of this study was to determine mRNA bcr-abl and the percentage of LSC in peripheral blood of patients with CML and ALL and identify a possible association between the amount of LSC in PB and clinical data. We included in this study 19 patients with Leukemia. A PB sample was collected per patient and leukocytes were obtained by Ficoll gradient. The immunophenotype for LSC CD34+ was done by flow cytometry analysis with CD33, CD2, CD14, CD16, CD64, HLA-DR, CD13, CD15, CD19, CD10, CD20, CD34, CD38, CD71, CD90, CD117, CD123 monoclonal antibodies. In addition, to identify the presence of the mRNA bcr-abl by RT-PCR, the RNA was isolated using TRIZOL reagent. Molecular (presence of mRNA bcr-abl and LSC CD34+) and clinical results were analyzed with descriptive statistics and a multiple regression analysis was performed to determine statistically significant association. In total, 19 patients (8 patients with ALL and 11 patients with CML) were analyzed, 9 patients with de novo leukemia (ALL = 6 and CML = 3) and 10 under treatment (ALL = 5 and CML = 5). The overall frequency of mRNA bcr-abl was 31% (6/19), and it was negative in ALL patients and positive in 80% in CML patients. On the other hand, LSC was determined in 16/19 leukemia patients (%LSC= 0.02-17.3). The Novo patients had higher percentage of LSC (0.26 to 17.3%) than patients under treatment (0 to 5.93%). The amount of LSC was significantly associated with the amount of LSC were: absence of treatment, the absence of splenomegaly, and a lower number of leukocytes, negative association for the clinical variables age, sex, blasts, and mRNA bcr-abl. In conclusion, patients with de novo leukemia had a higher percentage of circulating LSC than patients under treatment, and it was associated with clinical parameters as lack of treatment, absence of splenomegaly and a lower number of leukocytes. The mRNA bcr-abl detection was only possible in the series of patients with CML, and molecular detection of LSC could be identified in the peripheral blood of all leukemia patients, we believe the identification of circulating LSC may be used as biomarker for the detection of the MRD in leukemia patients.

Keywords: stem cells, leukemia, biomarkers, flow cytometry

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Authors: Pakize Neslihan Taslı, Alev Cumbul, Gul Merve Yalcın, Fikrettin Sahin

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A key experimental trial in the regeneration of large oral and craniofacial defects is the neogenesis of osseous and ligamentous interfacial structures. Currently, oral regenerative medicine strategies are unpredictable for repair of tooth supporting tissues destroyed as a consequence of trauma, chronic infection or surgical resection. A different approach combining the gel-casting method with Hydroxy Apatite HA-based scaffold and different cell lineages as a hybrid system leads to successively mimic the early stage of tooth development, in vitro. HA is widely accepted as a bioactive material for guided bone and tooth regeneration. In this study, it was reported that, HA porous scaffold preparation, characterization and evaluation of structural and chemical properties. HA is the main factor that exists in tooth and it is in harmony with structural, biological, and mechanical characteristics. Here, this study shows mimicking immature tooth at the late bell stage design and construction of HA scaffolds for cell transplantation of human Adipose Stem Cells (hASCs), human Bone Marrow Stem Cells (hBMSCs) and Gingival Epitelial cells for the formation of human tooth dentin-pulp-enamel complexes in vitro. Scaffold characterization was demonstrated by SEM, FTIR and pore size and density measurements. The biological contraction of dental tissues against each other was demonstrated by mRNA gene expressions, histopatologic observations and protein release profile by ELISA tecnique. The tooth shaped constructs with a pore size ranging from 150 to 300 µm arranged by gathering right amounts of materials provide interconnected macro-porous structure. The newly formed tissue like structures that grow and integrate within the HA designed constructs forming tooth cementum like tissue, pulp and bone structures. These findings are important as they emphasize the potential biological effect of the hybrid scaffold system. In conclusion, this in vitro study clearly demonstrates that designed 3D scaffolds shaped as a immature tooth at the late bell stage were essential to form enamel-dentin-pulp interfaces with an appropriate cell and biodegradable material combination. The biomimetic architecture achieved here is providing a promising platform for dental tissue engineering.

Keywords: tooth regeneration, tissue engineering, adipose stem cells, hydroxyapatite tooth engineering, porous scaffold

Procedia PDF Downloads 234

Authors: Nicole C. Valdez, Vincent L. Borromeo, Conrad C. Chong, Ahmad F. Mazahery

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Breast cancer is the most prevalent cancer worldwide, where the majority of cases are estrogen-receptor positive and involve 2 receptor proteins. The binding of estrogen to estrogen receptor alpha (ERα) promotes breast cancer growth, while it's binding to estrogen-receptor beta (ERβ) inhibits tumor growth. While natural products have been a promising source of chemotherapeutic agents, the challenge remains in finding a bioactive compound that specifically targets cancer cells, minimizing side effects on normal cells. Flavonoids are natural products that act as phytoestrogens and induce the same response as estrogen. They are able to compete with estrogen for binding to ERα; however, it has a higher binding affinity for ERβ. Their abundance in nature and low toxicity make them a potential candidate for breast cancer treatment. This study aimed to determine which particular flavonoids can specifically recognize ERβ and potentially be used for breast cancer treatment through molecular docking. A total of 206 flavonoids comprised of 97 isoflavones and 109 flavanones were collected from ZINC15, while the 3D structures of ERβ and ERα were obtained from Protein Data Bank. These flavonoid subclasses were chosen as they bind more strongly to ERs due to their chemical structure. The structures of the flavonoid ligands were converted using Open Babel, while the estrogen receptor protein structures were prepared using Autodock MGL Tools. The optimal binding site was found using BIOVIA Discovery Studio Visualizer before docking all flavonoids on both ERβ and ERα through Autodock Vina. Genistein is a flavonoid that exhibits anticancer effects by binding to ERβ, so its binding affinity was used as a baseline. Eriodictyol and 4”,6”-Di-O-Galloylprunin both exceeded genistein’s binding affinity for ERβ and was lower than its binding affinity for ERα. Of the two, eriodictyol was pursued due to its antitumor properties on a lung cancer cell line and on glioma cells. It is able to arrest the cell cycle at the G2/M phase by inhibiting the mTOR/PI3k/Akt cascade and is able to induce apoptosis via the PI3K/Akt/NF-kB pathway. Protein pathway and gene analysis were also conducted using ChEMBL and PANTHER and it was shown that eriodictyol might induce anticancer effects through the ROS1, CA7, KMO, and KDM1A genes which are involved in cell proliferation in breast cancer, non-small cell lung cancer, and other diseases. The high binding affinity of eriodictyol to ERβ, as well as its potential affected genes and antitumor effects, therefore, make it a candidate for the development of new breast cancer treatment. Verification through in vitro experiments such as checking the upregulation and downregulation of genes through qPCR and checking cell cycle arrest using a flow cytometry assay is recommended.

Keywords: breast cancer, estrogen receptor, flavonoid, molecular docking

Procedia PDF Downloads 89

Authors: Yakup Ulusu, Numan Eczacıoğlu, İsa Gökçe, Helen Waller, Jeremy H. Lakey

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Besides having the appropriate amino acid sequence to perform the function of proteins, it is important to have correct conformation after this sequence to process. To consist of this conformation depends on the amino acid sequence at the primary structure, hydrophobic interaction, chaperones and enzymes in charge of folding etc. Misfolded proteins are not functional and tend to be aggregated. Cysteine originating disulfide cross-links make stable this conformation of functional proteins. When two of the cysteine amino acids come side by side, disulfide bond is established that forms a cystine bridge. Due to this feature cysteine plays an important role on the formation of three-dimensional structure of many proteins. There are two cysteine amino acids (C44, C69) in the Tol-A-III protein. Unlike protein disulfide bonds from within his own, any non-specific cystine bridge causes a change in the three dimensional structure of the protein. Proteins can be expressed in various host cells as directly or fusion (chimeric). As a result of overproduction of the recombinant proteins, aggregation of insoluble proteins in the host cell can occur by forming a crystal structure called inclusion body. In general fusion proteins are produced for provide affinity tags to make proteins more soluble and production of some toxic proteins via fusion protein expression system like pTolT. Proteins can be modified by using a site-directed mutagenesis. By this way, creation of non-specific disulfide crosslinks can be prevented at fusion protein expression system via the present cysteine replaced by another amino acid such as serine, glycine or etc. To do this, we need; a DNA molecule that contains the gene that encodes for the target protein, required primers for mutation to be designed according to site directed mutagenesis reaction. This study was aimed to be replaced cysteine encoding codon TGT with serine encoding codon AGT. For this sense and reverse primers designed (given below) and used site-directed mutagenesis reaction. Several new copy of the template plasmid DNA has been formed with above mentioned mutagenic primers via polymerase chain reaction (PCR). PCR product consists of both the master template DNA (wild type) and the new DNA sequences containing mutations. Dpn-l endonuclease restriction enzyme which is specific for methylated DNA and cuts them to the elimination of the master template DNA. E. coli cells obtained after transformation were incubated LB medium with antibiotic. After purification of plasmid DNA from E. coli, the presence of the mutation was determined by DNA sequence analysis. Developed this new plasmid is called PtolT-δ.

Keywords: site directed mutagenesis, Escherichia coli, pTolT, protein expression

Procedia PDF Downloads 374

Authors: C. J. Tien, C. S. Chen, S. F. Huang, Z. X. Wang

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Algae in soil ecosystems can produce organic matters and oxygen by photosynthesis. Heterocyst-forming cyanobacteria can fix nitrogen to increase soil nitrogen contents. Secretion of mucilage by some algae increases the soil water content and soil aggregation. These actions will improve soil quality and fertility, and further increase abundance and diversity of soil microorganisms. In addition, some mixotrophic and heterotrophic algae are able to degrade petroleum hydrocarbons. Therefore, the objectives of this study were to analyze the effects of algal addition on the degradation of total petroleum hydrocarbons (TPH), diversity and activity of bacteria and algae in the diesel-contaminated soil under different nutrient contents and frequency of plowing and irrigation in order to assess the potential bioremediation technique using edaphic algae. The known amount of diesel was added into the farmland soil. This diesel-contaminated soil was subject to five settings, experiment-1 with algal addition by plowing and irrigation every two weeks, experiment-2 with algal addition by plowing and irrigation every four weeks, experiment-3 with algal and nutrient addition by plowing and irrigation every two weeks, experiment-4 with algal and nutrient addition by plowing and irrigation every four weeks, and the control without algal addition. Soil samples were taken every two weeks to analyze TPH concentrations, diversity of bacteria and algae, and catabolic genes encoding functional degrading enzymes. The results show that the TPH removal rates of five settings after the two-month experimental period were in the order: experiment-2 > expermient-4 > experiment-3 > experiment-1 > control. It indicated that algal addition enhanced the degradation of TPH in the diesel-contaminated soil, but not for nutrient addition. Plowing and irrigation every four weeks resulted in more TPH removal than that every two weeks. The banding patterns of denaturing gradient gel electrophoresis (DGGE) revealed an increase in diversity of bacteria and algae after algal addition. Three petroleum hydrocarbon-degrading algae (Anabaena sp., Oscillatoria sp. and Nostoc sp.) and two added algal strains (Leptolyngbya sp. and Synechococcus sp.) were sequenced from DGGE prominent bands. The four hydrocarbon-degrading bacteria Gordonia sp., Mycobacterium sp., Rodococcus sp. and Alcanivorax sp. were abundant in the treated soils. These results suggested that growth of indigenous bacteria and algae were improved after adding edaphic algae. Real-time polymerase chain reaction results showed that relative amounts of four catabolic genes encoding catechol 2, 3-dioxygenase, toluene monooxygenase, xylene monooxygenase and phenol monooxygenase were appeared and expressed in the treated soil. The addition of algae increased the expression of these genes at the end of experiments to biodegrade petroleum hydrocarbons. This study demonstrated that edaphic algae were suitable biomaterials for bioremediating diesel-contaminated soils with plowing and irrigation every four weeks.

Keywords: catabolic gene, diesel, diversity, edaphic algae

Procedia PDF Downloads 280

Authors: José Maria, Vânia Laranjo, Luís Abrunhosa, António Inês

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The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Keywords: carboxypeptidase, lactic acid bacteria, mycotoxins, ochratoxin a.

Procedia PDF Downloads 462

Authors: Karthick Dharmalingam, Stalin Ramakrishnan, Haseena Banu Hedayathullah Khan, Sachidanandanam Thiruvaiyaru Panchanadham, Shanthi Palanivelu

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Background: Breast cancer is arising as the most dreadful cancer affecting women worldwide. Hence, there arises a need to search and test for new drugs. Herbal formulations used in Siddha preparations are proved to be effective against various types of cancer. They also offer advantage through synergistic amplification and diminish any possible adverse effects. Tridham (TD) is a herbal formulation prepared in our laboratory consisting of Terminalia chebula, Elaeocarpus ganitrus and Prosopis cineraria in a definite ratio and has been used for the treatment of mammary carcinoma. Objective: To study the restorative effect of Tridham and penta galloyl glucose (a component of TD) on DMBA induced mammary carcinoma in female Sprague Dawley rats. Materials and Methods: Rats were divided into seven groups of six animals each. Group I (Control) received corn oil. Group II– mammary carcinoma was induced by DMBA dissolved in corn oil single dose orally. Group III and Group IV were induced with DMBA and subsequently treated with Tridham and penta galloyl glucose, respectively for 48 days. Group V was treated with DMBA and subsequently with a standard drug, cyclophosphamide. Group VI and Group VII were given Tridham and penta galloyl glucose alone, respectively for 48 days. After the experimental period, the animals were sacrificed by cervical decapitation. The mammary gland tissue was excised and levels of antioxidants were determined by biochemical assay. p53 and PCNA expression were accessed using immunohistochemistry. Nrf-2, Cox-2 and caspase-3 protein expression were studied by Western Blotting analysis. p21, Bcl-2, Bax, Bad and caspase-8 gene expression were studied by RT-PCR. Results: Histopathological studies confirmed induction of mammary carcinoma in DMBA induced rats and treatment with TD and PGG resulted in regression of tumour. The levels of enzymic and non-enzymic antioxidants were decreased in DMBA induced rats when compared to control rats. The levels of cell cycle inhibitory markers and apoptotic markers were decreased in DMBA induced rats when compared to control rats. These parameters were restored to near normal levels on treatment with Tridham and PGG. Conclusion: The results of the present study indicate the antineoplastic effect of Tridham and PGG are exerted through the modulation of antioxidant status and expression of cell cycle regulatory markers as well as apoptotic markers. Acknowledgment: Financial assistance provided in the form of ICMR-SRF by Indian Council of Medical Research (ICMR), India is gratefully acknowledged here.

Keywords: antioxidants, Mammary carcinoma, pentaGalloyl glucose, Tridham

Procedia PDF Downloads 279

Authors: Dace Grauda, Gunta Cekstere, Inta Belogrudova, Andis Karlsons, Isaak Rashal

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Trifolium repens L. (white or Dutch clover) is a perennial herb, belongs to legume family (Leguminosae Juss.), spread extensively by stolons and seeds. The species is cultivated worldwide and was naturalized in many countries in meadows, yards, gardens, along roads and streets etc., especially in temperate regions. It is widespread also in grasslands throughout Riga, the capital of Latvia. The goal of this study was to investigate genetic structure of white clover population in Riga and to evaluate influence of different salt concentration on plants. For this purpose universal retrotranspozone based IRAP (Inter-Retrotransposon Amplified Polymorphism) method was used. The plant material was collected in different regions of Riga and in several urban areas of Latvia. Plant DNA was isolated from in silicogel dried leaves of using 1% CTAB (cetyltrimet-ammonium bromide) buffer DNA extraction procedure. Genetic structure of city population and wild populations were compared. Soil salinization is an important issue associated with low water resources and highly urbanized areas in aride and semi-aride climate conditions, as well as de-icing salt application to prevent ice formation on roads in winter. The T. repens variety ‘Daile’ (form giganteum), one of the often used component of urban greeneries, was studied in this investigation. Plants were grown from seeds and cultivated in the light conditions (18-25 C, 16h/8h of day/night, light intensity 3000 lx) in plastic pots (200 ml), filled with commercial neutralized (pH 5.9 ± 0.3) peat substrate with mineral nutrients. To analyse the impact of increased soil salinity treatments with gradually rising NaCl (0; 20; 40; 60; 80; 100 mM) levels were arranged. Plants were watered when necessary with deionised water to provide optimum substrate moisture 60-70%. The experiment was terminated six weeks after establishment. For analysis of mineral nutrients, dry plant material (above ground part and roots) was used. Decrease of Na content can be significant under elevated salinity till 20 mM NaCl. High NaCl concentrations in the substrate increase Na, Cl, Cu, Fe, and Mn accumulation, but reduce S, Mg, K content in the plant above ground parts. Abiotic stresses generally changes the levels of DNA metilation. Several candidate gene for salt tolerance will be analysed for DNA metilation level using Pyromark-Q24 advanced.

Keywords: DNA metilation, IRAP, soil salinization, white clover

Procedia PDF Downloads 364

Authors: Otuwose E. Agyeno, Adeniyi A. Jayeola, Bashir A. Ajala

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P. esculentus is indigenous to Nigeria yet no wild relation has been encountered or reported. This has made it difficult to establish proper lineages between the varieties and landraces under cultivation. The present work is the first to determine the apormophy of 135 morphoanatomical characters in organs of 46 accessions drawn from 23 populations of this species based on dicta. The character states were coded in accession x character-state matrices and only 83 were informative and utilised for neighbour joining clustering based on euclidean values, and heuristic search in parsimony analysis using PAST ver. 3.15 software. Compatibility and evolutionary trends between accessions were then explored from values and diagrams produced. The low consistency indices (CI) recorded support monophyly and low homoplasy in this taxon. Agglomerative schedules based on character type and source data sets divided the accessions into mainly 3 clades, each of complexes of accessions. Solenostemon rotundifolius (Poir) J.K Morton was the outgroup (OG) used, and it occurred within the largest clades except when the characters were combined in a data set. The OG showed better compatibility with accessions of populations of landrace Isci, and varieties Riyum and Long’at. Otherwise, its aerial parts are more consistent with those of accessions of variety Bebot. The highly polytomous clades produced due to anatomical data set may be an indication of how stable such characters are in this species. Strict consensus trees with more than 60 nodes outputted showed that the basal nodes were strongly supported by 3 to 17 characters across the data sets, suggesting that populations of this species are more alike. The OG was clearly the first diverging lineage and closely related to accessions of landrace Gwe and variety Bebot morphologically, but different from them anatomically. It was also distantly related to landrace Fina and variety Long’at in terms of root, stem and leaf structural attributes. There were at least 5 other clades with each comprising of complexes of accessions from different localities and terrains within the study area. Spherical stem in cross section, size of vascular bundles at the stem corners as well as the alternate and whorl phyllotaxy are attributes which may have facilitated each other’s evolution in all accessions of the landrace Gwe, and they may be innovative since such states are not characteristic of the larger Lamiaceae, and Plectranthus L’Her in particular. In conclusion, this study has provided valuable information about infraspecific diversity in this taxon. It supports recognition of the varietal statuses accorded to populations of P. esculentus, as well as the hypothesis that the wild gene might have been distributed on the Jos Plateau. However, molecular characterisation of accessions of populations of this species would resolve this problem better.

Keywords: clustering, lineage, morphoanatomical characters, Nigeria, phylogenetics, Plectranthus esculentus, population

Procedia PDF Downloads 135

Authors: Khin Aye Myint, Mohd Rafii Yusop, Mohd Yusoff Abd Samad, Shairul Izan Ramlee, Mohd Din Amiruddin, Zulkifli Yaakub

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The narrow base of genetic is the main obstacle of breeding and genetic improvement in oil palm industry. In order to broaden the genetic bases, the Malaysian Palm Oil Board has been extensively collected wild germplasm from its original area of 11 African countries which are Nigeria, Senegal, Gambia, Guinea, Sierra Leone, Ghana, Cameroon, Zaire, Angola, Madagascar, and Tanzania. The germplasm collections were established and maintained as a field gene bank in Malaysian Palm Oil Board (MPOB) Research Station in Kluang, Johor, Malaysia to conserve a wide range of oil palm genetic resources for genetic improvement of Malaysian oil palm industry. Therefore, assessing the performance and genetic diversity of the wild materials is very important for understanding the genetic structure of natural oil palm population and to explore genetic resources. Principal component analysis (PCA) and Cluster analysis are very efficient multivariate tools in the evaluation of genetic variation of germplasm and have been applied in many crops. In this study, eight populations of MPOB-Senegal oil palm germplasm were studied to explore the genetic variation pattern using PCA and cluster analysis. A total of 20 yield and yield component traits were used to analyze PCA and Ward’s clustering using SAS 9.4 version software. The first four principal components which have eigenvalue >1 accounted for 93% of total variation with the value of 44%, 19%, 18% and 12% respectively for each principal component. PC1 showed highest positive correlation with fresh fruit bunch (0.315), bunch number (0.321), oil yield (0.317), kernel yield (0.326), total economic product (0.324), and total oil (0.324) while PC 2 has the largest positive association with oil to wet mesocarp (0.397) and oil to fruit (0.458). The oil palm population were grouped into four distinct clusters based on 20 evaluated traits, this imply that high genetic variation existed in among the germplasm. Cluster 1 contains two populations which are SEN 12 and SEN 10, while cluster 2 has only one population of SEN 3. Cluster 3 consists of three populations which are SEN 4, SEN 6, and SEN 7 while SEN 2 and SEN 5 were grouped in cluster 4. Cluster 4 showed the highest mean value of fresh fruit bunch, bunch number, oil yield, kernel yield, total economic product, and total oil and Cluster 1 was characterized by high oil to wet mesocarp, and oil to fruit. The desired traits that have the largest positive correlation on extracted PCs could be utilized for the improvement of oil palm breeding program. The populations from different clusters with the highest cluster means could be used for hybridization. The information from this study can be utilized for effective conservation and selection of the MPOB-Senegal oil palm germplasm for the future breeding program.

Keywords: cluster analysis, genetic variability, germplasm, oil palm, principal component analysis

Procedia PDF Downloads 164

Authors: I. Nava-Arenas, N. Ruiz-Ordaz, C. J. Galindez-Mayer, M. L. Luna-Guido, S. L. Ruiz-López, A. Cabrera-Orozco, D. Nava-Arenas

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Among the most important herbicides, the organochlorine compounds are of considerable interest due to their recalcitrance to the chemical, biological, and photolytic degradation, their persistence in the environment, their mobility, and their bioacummulation. The most widely used herbicides in North America are primarily 2,4-dichlorophenoxyacetic acid (2,4-D), the triazines (atrazine and simazine), and to a lesser extent diuron. The contamination of soils and water bodies frequently occurs by mixtures of these xenobiotics. For this reason, in this work, the operational stability to changes in the composition of the medium supplied to an aerobic biofilm reactor was studied. The reactor was packed with fragments of volcanic rock that retained a complex microbial film, able to degrade a mixture of organochlorine herbicides atrazine, simazine, diuron and 2,4-D, and whose members have microbial genes encoding the main catabolic enzymes atzABCD, tfdACD and puhB. To acclimate the attached microbial community, the biofilm reactor was fed continuously with a mineral minimal medium containing the herbicides (in mg•L-1): diuron, 20.4; atrazine, 14.2, simazine, 11.4, and 2,4-D, 59.7, as carbon and nitrogen sources. Throughout the bioprocess, removal efficiencies of 92-100% for herbicides, 78-90% for COD, 92-96% for TOC and 61-83% for dehalogenation were reached. In the microbial community, the genes encoding catabolic enzymes of different herbicides tfdACD, puhB and, occasionally, the genes atzA and atzC were detected. After the acclimatization, the triazine herbicides were eliminated from the mixture formulation. Volumetric loading rates of the mixture 2,4-D and diuron were continuously supplied to the reactor (1.9-21.5 mg herbicides •L-1 •h-1). Along the bioprocess, the removal efficiencies obtained were 86-100% for the mixture of herbicides, 63-94% for for COD, 90-100% for COT, and dehalogenation values of 63-100%. It was also observed that the genes encoding the enzymes in the catabolism of both herbicides, tfdACD and puhB, were consistently detected; and, occasionally, the atzA and atzC. Subsequently, the triazine herbicide atrazine and simazine were restored to the medium supply. Different volumetric charges of this mixture were continuously fed to the reactor (2.9 to 12.6 mg herbicides •L-1 •h-1). During this new treatment process, removal efficiencies of 65-95% for the mixture of herbicides, 63-92% for COD, 66-89% for TOC and 73-94% of dehalogenation were observed. In this last case, the genes tfdACD, puhB and atzABC encoding for the enzymes involved in the catabolism of the distinct herbicides were consistently detected. The atzD gene, encoding the cyanuric hydrolase enzyme, could not be detected, though it was determined that there was partial degradation of cyanuric acid. In general, the community in the biofilm reactor showed some catabolic stability, adapting to changes in loading rates and composition of the mixture of herbicides, and preserving their ability to degrade the four herbicides tested; although, there was a significant delay in the response time to recover to degradation of the herbicides.

Keywords: biodegradation, biofilm reactor, microbial community, organochlorine herbicides

Procedia PDF Downloads 435

Authors: C. Miranda, S. Cunha, R. Soares, M. Maia, G. Igrejas, F. Silva, P. Poeta

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The worldwide inappropriate use of antibiotics has increased the emergence of antimicrobial-resistant microorganisms isolated from animals, humans, food, and the environment. To combat this complex and multifaceted problem is essential to know the prevalence in livestock animals and possible ways of transmission among animals and between these and humans. Enterococci species, in particular E. faecalis and E. faecium, are the most common nosocomial bacteria, causing infections in animals and humans. Thus, the aim of this study was to characterize resistance and virulence factors genes among two enterococci species isolated from calf colostrums in Portuguese dairy farms. The 55 enterococci isolates (44 E. faecalis and 11 E. faecium) were tested for the presence of the resistance genes for the following antibiotics: erythromicyn (ermA, ermB, and ermC), tetracycline (tetL, tetM, tetK, and tetO), quinupristin/dalfopristin (vatD and vatE) and vancomycin (vanB). Of which, 25 isolates (15 E. faecalis and 10 E. faecium) were tested until now for 8 virulence factors genes (esp, ace, gelE, agg, cpd, cylA, cylB, and cylLL). The resistance and virulence genes were performed by PCR, using specific primers and conditions. Negative and positive controls were used in all PCR assays. All enterococci isolates showed resistance to erythromicyn and tetracycline through the presence of the genes: ermB (n=29, 53%), ermC (n=10, 18%), tetL (n=49, 89%), tetM (n=39, 71%) and tetK (n=33, 60%). Only two (4%) E. faecalis isolates showed the presence of tetO gene. No resistance genes for vancomycin were found. The virulence genes detected in both species were cpd (n=17, 68%), agg (n=16, 64%), ace (n=15, 60%), esp (n=13, 52%), gelE (n=13, 52%) and cylLL (n=8, 32%). In general, each isolate showed at least three virulence genes. In three E. faecalis isolates was not found virulence genes and only E. faecalis isolates showed virulence genes for cylA (n=4, 16%) and cylB (n=6, 24%). In conclusion, these colostrum samples that were consumed by calves demonstrated the presence of antibiotic-resistant enterococci harbored virulence genes. This genotypic characterization is crucial to control the antibiotic-resistant bacteria through the implementation of restricts measures safeguarding public health. Acknowledgements: This work was funded by the R&D Project CAREBIO2 (Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics - towards innovative theragnostic biomarkers), with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).

Keywords: antimicrobial resistance, calf, colostrums, enterococci

Procedia PDF Downloads 199

Authors: Alireza Barari, Ahmad Abdi

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Regular body trainings cause adaption in various system in body. One of the important effect of body training is its effect on immune system. It seems that cytokines usually release after long period exercises or some exercises which cause skeletal muscular damages. If some of the cytokines which cause responses such as inflammation of cells in skeletal muscles, with manipulating of training program, it can be avoided or limited from those exercises which induct cytokines release. Ginger plant is a kind of medicinal plants which is known as a anti inflammation plant. This plant is as most precedence medicinal plants in medicine science especially in inflammation cure. The aim of the present study was the effect of selected resistance training and consumption of ginger extract on IL-1α and TNFα untrained young women. The population includes young women interested in participating in the study with the average of 30±2 years old from Abbas Abad city among which 32 participants were chosen randomly and divided into 4 four groups, resistance training (R), resistance training and ginger consumption(RG), Ginger consumption(G)and Control group(C). The training groups performed circuit resistance training at the intensity of 65-75% one repeat maximum, 3 days a week for 6 weeks. Besides resistance training, subjects were given either ginseng (5 mg/kg per day) or placebo. Prior to and 48 hours after interventions body composition was measured and blood samples were taken in order to assess serum levels of IL-1α and TNFα. Plasma levels of cytokines were measured with commercially available ELISA Kits.IL-1α kit and TNFα kit were used in this research. To demonstrate the effectiveness of the independent variable and the comparison between groups, t-test and ANOVA were used. To determine differences between the groups, the Scheffe test was used that showed significant changes in any of the variables. we observed that circuit resistance training in R and RG groups can significant decreased in weight and body mass index in untrained females (p<0.05). The results showed a significant decreased in the mean level of IL-1α levels before and after the training period in G group (p=0.046) and RG group (p=0.022). Comparison between groups also showed there was significant difference between groups R-RG and RG-C. Intergroup comparison results showed that the mean levels of TNFα before and after the training in group G (p=0.044) and RG (p=0.037), significantly decreased. Comparison between groups also showed there was significant difference between groups R–RG , R-G ,RG-C and G-C. The research shows that circuit resistance training with reducing overload method results in systemic inflammation had significant effect on IL-1α levels and TNFα. Of course, Ginger can counteract the negative effects of resistance training exercise on immune function and stability of the mast cell membrane. Considerable evidence supported the anti-inflammatory properties of ginger for several constituents, especially gingerols, shogaols, paradols, and zingerones, through decreased cytokine gene TNF α and IL-1Α expression and inhibition of cyclooxygenase 1 and 2. These established biological actions suggest that ingested ginger could block the increase in IL-1α.

Keywords: resistance training, ginger, IL-1α , TNFα

Procedia PDF Downloads 428

Authors: S. D. N. K. Bathige, G. I. Godahewa, Navaneethaiyer Umasuthan, Jehee Lee

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Kunitz-type serine protease inhibitors (KTIs) are identified in various organisms including animals, plants and microbes. These proteins shared single or multiple Kunitz inhibitory domains link together or associated with other types of domains. Characteristic Kunitz type domain composed of around 60 amino acid residues with six conserved cysteine residues to stabilize by three disulfide bridges. KTIs are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis and inflammation. In this study, two Kunitz-type domain containing protein was identified from rock bream database and designated as RbKunitz. The coding sequence of RbKunitz encoded for 507 amino acids with 56.2 kDa theoretical molecular mass and 5.7 isoelectric point (pI). There are several functional domains including MANEC superfamily domain, PKD superfamily domain, and LDLa domain were predicted in addition to the two characteristic Kunitz domain. Moreover, trypsin interaction sites were also identified in Kunitz domain. Homology analysis revealed that RbKunitz shared highest identity (77.6%) with Takifugu rubripes. Completely conserved 28 cysteine residues were recognized, when comparison of RbKunitz with other orthologs from different taxonomical groups. These structural evidences indicate the rigidity of RbKunitz folding structure to achieve the proper function. The phylogenetic tree was constructed using neighbor-joining method and exhibited that the KTIs from fish and non-fish has been evolved in separately. Rock bream was clustered with Takifugu rubripes. The SYBR Green qPCR was performed to quantify the RbKunitz transcripts in different tissues and challenged tissues. The mRNA transcripts of RbKunitz were detected in all tissues (muscle, spleen, head kidney, blood, heart, skin, liver, intestine, kidney and gills) analyzed and highest transcripts level was detected in gill tissues. Temporal transcription profile of RbKunitz in rock bream blood tissues was analyzed upon LPS (lipopolysaccharide), Poly I:C (Polyinosinic:polycytidylic acid) and Edwardsiella tarda challenge to understand the immune responses of this gene. Compare to the unchallenged control RbKunitz exhibited strong up-regulation at 24 h post injection (p.i.) after LPS and E. tarda injection. Comparatively robust expression of RbKunits was observed at 3 h p.i. upon Poly I:C challenge. Taken together all these data indicate that RbKunitz may involve into to immune responses upon pathogenic stress, in order to protect the rock bream.

Keywords: Kunitz-type, rock bream, immune response, serine protease inhibitor

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Authors: Adamu Usman, Grace Stanley Balami

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Pearl millet (Pennisetum glaucum L. R. Br.) is a cereal cultivated in arid and semi-arid areas of the world. It supports more than 100 million people around the world. Parasitic weed (Striga hermonthica Del. Benth) is a major constraint to its production. Estimated yield losses are put at 10 - 95% depending on variety, ecology and cultural practices. Potentials in selection of traits in pearl millets for grain yield have been reported and it depends on genotypic variability and heritability among landraces. Variability and heritability among cultivars could offer opportunities for improvement. The study was conducted to determine the genetic variability among cultivars and estimate broad sense heritability among grain yield and related traits. F1 breeding populations were generated with 9 parental cultivars, viz; Ex-Gubio, Ex-Monguno, Ex-Baga as males and PEO 5984, Super-SOSAT, SOSAT-C88, Ex-Borno and LCIC9702 as females through Line × Tester mating during 2017 dry season at Lushi Irrigation Station, Bauchi Metropolitan in Bauchi State, Nigeria. The F1 population and the parents were evaluated during cropping season of 2018 at Bauchi and Maiduguri. Data collected were subjected to analysis of variance. Results showed significant difference among cultivars and among traits indicating variability. Number of plants at emergence, days to 50% flowering, days to 100% flowering, plant height, panicle length, number of plants at harvest, Striga count at 90 days after sowing, panicle weight and grain yield were significantly different. Significant variability offer opportunity for improvement as superior individuals can be isolated. Genotypic variance estimates of traits were largely greater than environmental variances except in plant height and 1000 seed weight. Environmental variances were low and in some cases negligible. The phenotypic variances of all traits were higher than genotypic variances. Similarly phenotypic coefficient of variation (PCV) was higher than genotypic coefficient of variation (GCV). High heritability was found in days to 50% flowering (90.27%), Striga count at 90 days after sowing (90.07%), number of plants at harvest (87.97%), days to 100% flowering (83.89%), number of plants at emergence (82.19%) and plant height (73.18%). Greater heritability estimates could be due to presence of additive gene. The result revealed wider variability among genotypes and traits. Traits having high heritability could easily respond to selection. High value of GCV, PCV and heritability estimates indicate that selection for these traits are possible and could be effective.

Keywords: variability, heritability, phenotypic, genotypic, striga

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

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Drug delivery systems are proposed for use in cancer treatment to specifically target cancer cells and deliver a therapeutic dose without affecting normal cells. For that purpose, the use of folate receptors (FR) can be considered a key strategy, since they are commonly over-expressed in cancer cells. In this study, cyclodextrins (CD) have being used as vehicles to target FR and deliver the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within their cavities. Here, β-CD has been modified using folic acid so as to specifically target the FR. Thus, this drug delivery system consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 15.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 16.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 10.5 for A549 and 132.6 µM ± 16.1 and 288.1 µM ± 26.3 for BEAS-2B. These results demonstrate that free MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug. The use of cell imaging by confocal microscopy has allowed visualisation of FR targeting in cancer cells, as well as the identification of the interlisation pathway of the drug. Hence, the cellular uptake and internalisation process of this drug delivery system is being addressed.

Keywords: cancer treatment, cyclodextrins, drug delivery, folate receptors, reduced folate carriers

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Authors: Helen J. Spray, Karina J. Linnell

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Local-global bias in adulthood is strongly dependent on environmental factors and a global bias is not the universal characteristic of adult perception it was once thought to be: whilst Western adults typically demonstrate a global bias, Namibian adults living in traditional villages possess a strong local bias. Furthermore, environmental effects on local-global bias have been shown to be highly gender-specific; whereas urbanisation promoted a global bias in urbanised Namibian women but not men, education promoted a global bias in urbanised Namibian men but not women. Adult populations, however, provide only a snapshot of the gene-environment interactions which shape perceptual bias. Yet, to date, there has been little work on the development of local-global bias across environmental settings. In the current study, local-global bias was assessed using a similarity-matching task with Navon figures in children aged between 4 and 15 years from across three populations: traditional Namibians, urban Namibians, and urban British. For the two Namibian groups, measures of urbanisation and education were obtained. Data were subjected to both between-group and within-group analyses. Between-group analyses compared developmental trajectories across population and gender. These analyses revealed a global bias from even as early as 4 in the British sample, and showed that the developmental onset of a global bias is not fixed. Urbanised Namibian children ultimately developed a global bias that was indistinguishable from British children; however, a global bias did not emerge until much later in development. For all populations, the greatest developmental effects were observed directly following the onset of formal education. No overall gender effects were observed; however, there was a significant gender by age interaction which was difficult to reconcile with existing biological-level accounts of gender differences in the development of local-global bias. Within-group analyses compared the effects of urbanisation and education on local-global bias for traditional and urban Namibian boys and girls separately. For both traditional and urban boys, education mediated all effects of age and urbanisation; however, this was not the case for girls. Traditional Namibian girls retained a local bias regardless of age, education, or urbanisation, and in urbanised girls, the development of a global bias was not attributable to any one factor specifically. These results are broadly consistent with aforementioned findings that education promoted a global bias in urbanised Namibian men but not women. The development of local-global bias does not follow a fixed trajectory but is subject to environmental control. Understanding how variability in the development of local-global bias might arise, particularly in the context of gender, may have far-reaching implications. For example, a number of educationally important cognitive functions (e.g., spatial ability) are known to show consistent gender differences in childhood and local-global bias may mediate some of these effects. With education becoming an increasingly prevalent force across much of the developing world it will be important to understand the processes that underpin its effects and their implications.

Keywords: cross-cultural, development, education, gender, local-global bias, perception, urbanisation, urbanization

Procedia PDF Downloads 139

Authors: Nima Samie, Sekaran Muniandy, Zahurin Mohamed, M. S. Kanthimathi

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Although number of indole containing compounds have been reported to have anticancer properties in vitro but only a few of them show potential as anticancer compounds in vivo. The current study was to evaluate the mechanism of cytotoxicity of selected indolic compound in vivo and in vitro. In this context, we determined the potency of the compound in the induction of apoptosis, cell cycle arrest, and cytoskeleton rearrangement. HT-29, WiDr, CCD-18Co, human monocyte/macrophage CRL-9855, and B lymphocyte CCL-156 cell lines were used to determine the IC50 of the compound using the MTT assay. Analysis of apoptosis was carried out using immunofluorescence, acridine orange/ propidium iodide double staining, Annexin-V-FITC assay, evaluation of the translocation of NF-kB, oxygen radical antioxidant capacity, quenching of reactive oxygen species content, measurement of LDH release, caspase-3/-7, -8 and -9 assays and western blotting. The cell cycle arrest was examined using flowcytometry and gene expression was assessed using qPCR array. Results displayed a potent suppressive effect on HT-29 and WiDr after 24 h of treatment with IC50 value of 2.52±0.34 µg/ml and 2.13±0.65 µg/ml respectively. This cytotoxic effect on normal, monocyte/macrophage and B-cells was insignificant. Dipping in the mitochondrial membrane potential and increased release of cytochrome c from the mitochondria indicated induction of the intrinsic apoptosis pathway by the compound. Activation of this pathway was further evidenced by significant activation of caspase-9 and 3/7. The compound was also shown to activate the extrinsic pathways of apoptosis via activation of caspase-8 which is linked to the suppression of NF-kB translocation to the nucleus. Cell cycle arrest in the G1 phase and up-regulation of glutathione reductase, based on excessive ROS production were also observed. These findings were further investigated for inhibitory efficiency of the compound on colonic aberrant crypt foci in male rats. Rats were divided in to 5 groups: vehicle, cancer control, positive control groups and the groups treated with 25 and 50 mg/kg of compounds for 10 weeks. Administration of compound suppressed total colonic ACF formation up to 73.4%. The results also showed that treatment with the compound significantly reduced the level of malondialdehyde while increasing superoxide dismutase and catalase activities. Furthermore, the down-regulation of PCNA and Bcl2 and the up-regulation of Bax was confirmed by immunohistochemical staining. The outcome of this study suggest sthat the indolic compound is a potent anti-cancer agent against colon cancer and can be further evaluated by animal trial.

Keywords: indolic compound, chemoprevention, crypt, azoxymethane, colon cancer

Procedia PDF Downloads 348

Authors: Maria João Ferreira, Natalia Sierra-Garcia, Javier Cremades, Carla António, Ana M. Rodrigues, Helena Silva, Ângela Cunha

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Salicornia ramosissima, a halophyte that grows naturally in coastal areas of the northern hemisphere, is often considered the most promising halophyte candidate for extensive crop cultivation and saline agriculture practices. The expanding interest in this plant surpasses its use as gourmet food and includes their potential application as a source of bioactive compounds for the pharmaceutical industry. Despite growing well in saline soils, sustainable and ecologically friendly techniques to enhance crop production and the nutritional value of this plant are still needed. The root microbiome of S. ramosissima proved to be a source of taxonomically diverse plant growth-promoting bacteria (PGPB). Halotolerant strains of Bacillus, Salinicola, Pseudomonas, and Brevibacterium, among other genera, exhibit a broad spectrum of plant-growth promotion traits [e.g., 3-indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophores, phosphate solubilization, Nitrogen fixation] and express a wide range of extracellular enzyme activities. In this work, three plant growth-promoting bacteria strains (Brevibacterium casei EB3, Pseudomonas oryzihabitans RL18, and Bacillus aryabhattai SP20) isolated from the rhizosphere and the endosphere of S. ramosissima roots from different saltmarshes along the Portuguese coast were inoculated in S. ramosissima seeds. Plants germinated from inoculated seeds were grown for three months in pots filled with a mixture of perlite and estuarine sediment (1:1) in greenhouse conditions and later transferred to a growth chamber, where they were maintained two months with controlled photoperiod, temperature, and humidity. Pots were placed on trays containing the irrigation solution (Hoagland’s solution 20% added with 10‰ marine salt). Before reaching the flowering stage, plants were collected, and the fresh and dry weight of aerial parts was determined. Non-inoculated seeds were used as a negative control. Selected dried stems from the most promising treatments were later analyzed by GC-TOF-MS for primary metabolite composition. The efficiency of inoculation and persistence of the inoculum was assessed by Next Generation Sequencing. Inoculations with single strain EB3 and co-inoculations with EB3+RL18 and EB3+RL18+SP20 (All treatment) resulted in significantly higher biomass production (fresh and dry weight) compared to non-inoculated plants. Considering fresh weight alone, inoculation with isolates SP20 and RL18 also caused a significant positive effect. Combined inoculation with the consortia SP20+EB3 or SP20+RL18 did not significantly improve biomass production. The analysis of the profile of primary metabolites will provide clues on the mechanisms by which the growth-enhancement effect of the inoculants operates in the plants. These results sustain promising prospects for the use of rhizospheric and endophytic PGPB as biofertilizers, reducing environmental impacts and operational costs of agrochemicals and contributing to the sustainability and cost-effectiveness of saline agriculture. Acknowledgments: This work was supported by project Rhizomis PTDC/BIA-MIC/29736/2017 financed by Fundação para a Ciência e Tecnologia (FCT) through the Regional Operational Program of the Center (02/SAICT/2017) with FEDER funds (European Regional Development Fund, FNR, and OE) and by FCT through CESAM (UIDP/50017/2020 + UIDB/50017/2020), LAQV-REQUIMTE (UIDB/50006/2020). We also acknowledge FCT/FSE for the financial support to Maria João Ferreira through a PhD grant (PD/BD/150363/2019). We are grateful to Horta dos Peixinhos for their help and support during sampling and seed collection. We also thank Glória Pinto for her collaboration providing us the use of the growth chambers during the final months of the experiment and Enrique Mateos-Naranjo and Jennifer Mesa-Marín of the Departamento de Biología Vegetal y Ecología, the University of Sevilla for their advice regarding the growth of salicornia plants in greenhouse conditions.

Keywords: halophytes, PGPB, rhizosphere engineering, biofertilizers, primary metabolite profiling, plant inoculation, Salicornia ramosissima

Procedia PDF Downloads 160