Search results for: tumor suppressor gene

Authors: Akhmadjon Sultanov, Eun-Ho Lee, Hye-Jin Park, Young-Je Cho

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This study tested the anti-inflammatory effect of wild indigo (Baptisia tinctoria) root in Raw 264.7 cells. We prepared two extracts of B. tinctoria; one in water and the other in 50% ethanol. Then we evaluated the toxicities of the B. tinctoria root extracts at 10 to 100 mg mL-1 concentrations in raw 264.7 cells and observed 80% cell viability. The anti-inflammatory effect of B. tinctoria root extract in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells were observed with concentrations at 10, 30, and 50 μg mL-1. The results showed that 77.27-66.82% of nitric oxide (NO) production was inhibited by 50 μg mL-1 B. tinctoria root extract. The protein expression of Inducible NO synthase (iNOS) expression dramatically decreased by 93.14% and 52.65% in raw 264.7 cells treated with water and ethanol extracts of B. tinctoria root, respectively. Moreover, cyclooxygenase-2 (COX-2) protein expression decreased by 42.85% and 69.70% in raw 264.7 cells treated with water and ethanol extracts of B. tinctoria root, respectively. Furthermore, the mRNA expression of pro-inflammatory markers, such as tumor necrosis factor-alpha, interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, and prostaglandin E synthase 2, was significantly suppressed in a concentration-dependent manner. Additionally, the B. tinctoria root extracts effectively inhibited enzymes involved in physiological activities. The B. tinctoria root extracts showed excellent anti-inflammatory effects and can be used as a functional material for biological activities.

Keywords: cytokine, macrophage, pro-inflammatory, protein expression, real-time PCR

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Authors: Delly C. Lestari, Linosefa, Ardiana Kusumaningrum, Andi Yasmon, Anis Karuniawati

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The objective of this study is to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) harboring mecA genes from screening isolates among intensive care unit (ICU) patients. All MRSA screening isolates from ICU’s patients of Cipto Mangunkusumo Hospital during 2011 and 2014 were included in this study. Identification and susceptibility test was performed using Vitek2 system (Biomereux®). PCR was conducted to characterize the SCCmec of S. aureus harboring the mecA gene on each isolate. Patient’s history of illness was traced through medical record. 24 isolates from 327 screening isolates were MRSA positive (7.3%). From PCR, we found 17 (70.8%) isolates carrying SCCmec type I, 3 (12.5%) isolates carrying SCCmec type III, and 2 (8.3%) isolates carrying SCCmec type IV. In conclusion, SCCmec type I is the most prevalent MRSA colonization among ICU patients in Cipto Mangunkusumo Hospital.

Keywords: MRSA, mecA genes, ICU, colonization

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Authors: Artem Kim, Clara Savary, Christele Dubourg, Wilfrid Carre, Houda Hamdi-Roze, Valerie Dupé, Sylvie Odent, Marie De Tayrac, Veronique David

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Holoprosencephaly (HPE) is a rare congenital brain malformation resulting from the incomplete separation of the two cerebral hemispheres. It is characterized by a wide phenotypic spectrum and a high degree of locus heterogeneity. Genetic defects in 16 genes have already been implicated in HPE, but account for only 30% of cases, suggesting that a large part of genetic factors remains to be discovered. HPE has been recently redefined as a complex multigenic disorder, requiring the joint effect of multiple mutational events in genes belonging to one or several developmental pathways. The onset of HPE may result from accumulation of the effects of multiple rare variants in functionally-related genes, each conferring a moderate increase in the risk of HPE onset. In order to decipher the genetic basis of HPE, unconventional patterns of inheritance involving multiple genetic factors need to be considered. The primary objective of this study was to uncover possible disease causing combinations of multiple rare variants underlying HPE by performing trio-based Whole Exome Sequencing (WES) of familial cases where no molecular diagnosis could be established. 39 families were selected with no fully-penetrant causal mutation in known HPE gene, no chromosomic aberrations/copy number variants and without any implication of environmental factors. As the main challenge was to identify disease-related variants among a large number of nonpathogenic polymorphisms detected by WES classical scheme, a novel variant prioritization approach was established. It combined WES filtering with complementary gene-level approaches: transcriptome-driven (RNA-Seq data) and clinically-driven (public clinical data) strategies. Briefly, a filtering approach was performed to select variants compatible with disease segregation, population frequency and pathogenicity prediction to identify an exhaustive list of rare deleterious variants. The exome search space was then reduced by restricting the analysis to candidate genes identified by either transcriptome-driven strategy (genes sharing highly similar expression patterns with known HPE genes during cerebral development) or clinically-driven strategy (genes associated to phenotypes of interest overlapping with HPE). Deeper analyses of candidate variants were then performed on a family-by-family basis. These included the exploration of clinical information, expression studies, variant characteristics, recurrence of mutated genes and available biological knowledge. A novel bioinformatics pipeline was designed. Applied to the 39 families, this final integrated workflow identified an average of 11 candidate variants per family. Most of candidate variants were inherited from asymptomatic parents suggesting a multigenic inheritance pattern requiring the association of multiple mutational events. The manual analysis highlighted 5 new strong HPE candidate genes showing recurrences in distinct families. Functional validations of these genes are foreseen.

Keywords: complex genetic disorder, holoprosencephaly, multiple rare variants, whole exome sequencing

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Authors: Apotiola Z. O., Anyakorah C. I., Kuforiji O. O.

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Sensory and microbial properties of fresh and canned Calocybe indica (milky mushroom) were evaluated. The mushroom was grown under a controlled environment with hardwood (Cola nitida) and rice bran substrate (4:1) canned in a brine solution of salt and citric acid. Analysis was carried out using standard methods. The overall acceptability ranged between 5.62 and 6.50, with sample S30 adjudged the best. In all, significant differences p<0.01 exist in the panelist judgment. Thus, the incorporation of salt and citric acid at 3.5g and 1.5g, respectively, improved sensory attributes such as texture, aroma, color, and overall acceptability. There was no coliform and fungi growth on the samples throughout the storage period. The bacterial count, on the other hand, was observed only in the fifth and sixth week of the storage period which varied between 0.2 to 0.9 x 103 cfu/g. The highest value was observed in sample S20 of the sixth week of storage, while the lowest value was recorded in sample S30 of the sixth week of storage. Based on 16S rRNA gene sequencing, bacterial species were taxonomically confirmed as Bacillus thuringiensis. The percentile compositions and Sequence ID of the bacterial species in the mushroom was 90%.

Keywords: bacterial count, microbial property, sensory, sawdust, texture

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Authors: Manal Farea, Adam Husein, Ahmad S. Halim, Zurairah Berahim, Nurul A. Abdullah, Khairani I. Mokhtar, Kasmawati Mokhtar

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Endodontic furcal perforation remains both an endodontic and a periodontal problem. Regeneration of cementum is very essential for the perforation repair. The aim of this study was to investigate the role of Hertwig's epithelial root sheath (HERS) cells on the cementogenic differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) in the presence of chitosan scaffold-TGFβ1. HERS cells were isolated and characterized then co-cultured with SHED with/without chitosan scaffold-TGFβ1. SHED proliferation was assessed by PrestoBlue. Alkaline phosphatase activity, mineralization behaviour and gene/protein expression of cemento/osteoblast phenotype of SHED were evaluated. Results of the present study showed that HERS cells in association with chitosan-TGFβ1 enhanced proliferation and cemento/osteogenic differentiation of SHED. Our novel co-culture system confirmed the potential effect of HERS cells to stimulate the differentiation of SHED along the cementoblastic lineage which was triggered in the presence of chitosan-TGFβ1. This approach possesses a novel therapeutic strategy for future endodontic perforation and periodontitis.

Keywords: cementogenesis, co-culture system, HERS, SHED

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Authors: Hae-Jun Lee, Ji-Sun Shin, Kyung-Tae Lee

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Potentilla species (Rosasease) have been used in traditional medicine to treat different ailment, disease or malady. In this study, we investigated the anti-inflammatory effects of ethanol extracts of NUTT (EPP) in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages and septic mice. EPP suppressed LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-induced Raw 264.7 macrophages. Consistent with these observations, EPP reduced the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by downregulation of their promoter activities. EPP inhibited tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) at production and mRNA levels. Molecularly, EPP attenuated the LPS-induced transcriptional activity, and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α (IκB-α) degradation and IκB kinase-α/β (IKK-α/β) phosphorylation. Furthermore, EPP suppressed the LPS-induced activation of activator protein-1 (AP-1) by reducing the expression of c-Fos and c-Jun in nuclear. EPP also reduced the phosphorylation of mitogen-activated protein kinase (MAPK), such as p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). In a sepsis model, pretreatment with EPP reduced the LPS-induced lethality. Collectively, these results suggest that the anti-inflammatory effects of EPP were associated with the suppression of NF-κB and AP-1 activation, and support its possible therapeutic role for the treatment of sepsis.

Keywords: anti-inflammation, activator protein-1, nuclear factor κB, Potentilla paradoxa Nutt

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Authors: Corina Muscurel, Irina Stoian, Laura Gaman, Valeriu Atanasiu

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Chronic inflammation predisposes cells to neoplastic transformation and is associated with angiogenesis. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) give rise to anti-inflammatory metabolites and decrease some inflammatory cytokines. The aim of the study was to analyze the effect of n-3 PUFAs intake on patients with tumors in early-stage (without regional or distant metastasis). There were two groups of patients: one group with colon tumors and one group with lung tumors. All patients took for 60 days daily supplements from fish-oil containing 600 mg eicosapentaenoic acid and 400 mg docosahexaenoic acid. The plasma markers were evaluated before and after PUFAs intake: ceruloplasmin (using p-phenylenediamine oxidase method), plasma total thiol groups (using dithiobis-nitrobenzoic acid method) and CEA (carcinoembryonic antigen using electrochemiluminescent immunoassay). The results reflect ceruloplasmin decrease (p < 0.05), plasma total thiol groups increase (not statistically significant) and CEA decrease (p < 0.05) after n-3 PUFAs intake. Conclusions: n-3 PUFAs intake is favorable in premalignant lesions or in early tumor stage and dietary fish-oil has anti-inflammatory effects and can contribute to reduce cancer progression.

Keywords: cancer, fish-oil, inflammation, n-3 polyunsaturated fatty acids

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Authors: Taleb L., Benarbia M., Brahmi M., Belmiloud H., Boukerche A.

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Introduction and purpose of the study: Cavum cancer in children and adolescents is rare and represents 8% of all nasopharyngeal cancers treated in our department. Our objective is to study its epidemiological, clinical, therapeutic, and evolutionary particularities. Material and methods: Retrospective study of 39 patients under 20 years old, treated for undifferentiated non-metastatic carcinoma of the nasopharynx at the Emir Abdelkader Cancer Center between 2014 and 2020. Results and statistical analysis: Median age was 14 years [7-19 years], with a sex ratio of 2.9. The median time to diagnosis was 5.6 months [1 to 14 months], the circumstances of the discovery of which were dominated by lymph node syndrome in 43.6% of cases (n=17) followed by a rhinological syndrome in 30.8% of cases (n=13). The tumor stage was T1 for two patients (5.1%), T2 for 8 (20.5%), T3 for 9 (23.1%), T4 for 20 (51.3%), N0 for 2 (5 .1%) N1 for 4 (10.3%), N2 for 28 (71.8%) and N3 for 5 (12.8%). All patients received induction chemotherapy followed by concomitant radiotherapy with cisplatin. The dose of irradiation delivered to the cavum and adenopathies was 66 Gy with fractionation of 2 Gy per session in 69.2% of cases (n=27) and 1.8 Gy in 30.8% of cases (n=12). With a median follow-up of 51 months (15 to 97 months), the locoregional, metastatic, specific, and overall relapse-free survival rates at five years were 91.1%, 73.5%, 66.1%, and 68.4, respectively. Conclusion: Chemotherapy and radiotherapy treatment of cavum cancer in children and adolescents has allowed excellent locoregional control despite the advanced stage of the disease. However, the frequency of metastatic relapses could justify the possible use of systemic maintenance treatment.

Keywords: cancer, nasopharynx, radiotherapy, chemotherapy, survival

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Authors: M. N. Hanina, M. Hairul Shahril, I. Ismatul Nurul Asyikin, A. K. Abdul Jalil, M. R. Salina, M. R. Maryam, M. Rosfarizan

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The extracellular proteins secreted by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was carried out to determine the effect of Streptomycin Sulfate in regulating extracellular proteins secreted by Bacillus subtilis ATCC21332. Results of Microdilution assay showed that the Minimum Inhibition Concentration (MIC) of Streptomycin Sulfate on B. subtilis ATCC21332 was 2.5 mg/ml. The bacteria cells were then exposed to Streptomycin Sulfate at concentration of 0.01 MIC before being further incubated for 48h to 72 h. The extracellular proteins secreted were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile revealed that three additional bands with approximate sizes of 30 kDa, 22 kDa and 23 kDa were appeared for the treated bacteria with Streptomycin Sulfate. Thus, B. subtilis ATCC21332 in stressful condition with the presence of Streptomycin Sulfate at low concentration could induce the extracellular proteins secretion.

Keywords: Bacillus subtilis ATCC21332, streptomycin sulfate, extracellular proteins, antibiotics

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Authors: Osama Shakeel

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The objective of the paper is to study the clinic-pathological factors, survival analyses, recurrence rate, metastatic rate, risk factors and the management of cutaneous malignant melanoma at Shaukat Khanum Memorial Cancer Hospital and Research Center. Methodology: From 2014 to 2017, all patients with a diagnosis of cutaneous malignant melanoma (CMM) were included in the study. Demographic variables were collected. Short and long term oncological outcomes were recorded. All data were entered and analyzed in SPSS version 21. Results: A total of 28 patients were included in the study. Median age was 46.5 +/-15.9 years. There were 16 male and 12 female patients. The family history of melanoma was present in 7.1% (n=2) of the patients. All patients had a mean survival of 13.43+/- 9.09 months. Lower limb was the commonest site among all which constitutes 46.4%(n=13). On histopathological analyses, ulceration was seen in 53.6% (n=15) patients. Unclassified tumor type was present in 75%(n=21) of the patients followed by nodular 21.4% (n=6) and superficial spreading 3.5%(n=1). Clark level IV was the commonest presentation constituting 46.4%(n=13). Metastases were seen in 50%(n=14) of the patients. Local recurrence was observed in 60.7%(n=17). 64.3%(n=18) lived after one year of treatment. Conclusion: CMM is a fatal disease. Although its disease of fair skin individuals, however, the incidence of CMM is also rising in this part of the world. Management includes early diagnoses and prompt management. However, mortality associated with this disease is still not favorable.

Keywords: malignant cancer of skin, cutaneous malignant melanoma, skin cancer, survival analyses

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Authors: Khiem M. Nguyen, Ming C. Yang

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Chloroplast pigments are extremely important during photosynthesis since they play essential roles in light absorption and energy transfer. Therefore, understanding the efficiency of chlorophyll (Chl) biosynthesis could facilitate enhancement in photo-assimilates accumulation, and ultimately, in crop yield. The Chl-deficient mutants have been used extensively to study the Chl biosynthetic pathways and the biogenesis of the photosynthetic apparatus. Rice (Oryza sativa L.) is one of the most leading food crops, serving as staple food for many parts of the world. To author’s best knowledge, Chl b–lacking rice has been found; however the molecular mechanism of Chl biosynthesis still remains unclear compared to wild-type rice. In this study, the ultrastructure analysis, photosynthetic properties, and transcriptome profile of wild-type rice (Norin No.8, N8) and its Chl b-lacking mutant (Chlorina 1, C1) were examined. The finding concluded that total Chl content and Chl b content in the C1 leaves were strongly reduced compared to N8 leaves, suggesting that reduction in the total Chl content contributes to leaf color variation at the physiological level. Plastid ultrastructure of C1 possessed abnormal thylakoid membranes with loss of starch granule, large number of vesicles, and numerous plastoglobuli. The C1 rice also exhibited thinner stacked grana, which was caused by a reduction in the number of thylakoid membranes per granum. Thus, the different Chl a/b ratio of C1 may reflect the abnormal plastid development and function. Transcriptional analysis identified 23 differentially expressed genes (DEGs) and 671 transcription factors (TFs) that were involved in Chl metabolism, chloroplast development, cell division, and photosynthesis. The transcriptome profile and DEGs revealed that the gene encoding PsbR (PSII core protein) was down-regulated, therefore suggesting that the lower in light-harvesting complex proteins are responsible for the lower photosynthetic capacity in C1. In addition, expression level of cell division protein (FtsZ) genes were significantly reduced in C1, causing chloroplast division defect. A total of 19 DEGs were identified based on KEGG pathway assignment involving Chl biosynthesis pathway. Among these DEGs, the GluTR gene was down-regulated, whereas the UROD, CPOX, and MgCH genes were up-regulated. Observation through qPCR suggested that later stages of Chl biosynthesis were enhanced in C1, whereas the early stages were inhibited. Plastid structure analysis together with transcriptomic analysis suggested that the Chl a/b ratio was amplified both by the reduction in Chl contents accumulation, owning to abnormal chloroplast development, and by the enhanced conversion of Chl b to Chl a. Moreover, the results indicated the same Chl-cycle pattern in the wild-type and C1 rice, indicating another Chl b degradation pathway. Furthermore, the results demonstrated that normal grana stacking, along with the absence of Chl b and greatly reduced levels of Chl a in C1, provide evidence to support the conclusion that other factors along with LHCII proteins are involved in grana stacking. The findings of this study provide insight into the molecular mechanisms that underlie different Chl a/b ratios in rice.

Keywords: Chl-deficient mutant, grana stacked, photosynthesis, RNA-Seq, transcriptomic analysis

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Authors: N. A. Kazemi Sefat, M. M. Mohammadi, J. Hadjati, S. Talebi, M. Ajami, H. Daneshvar

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Colorectal cancer metastases result in a significant number of cancer related deaths. Histone deacetylase (HDAC) inhibitors induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (SB) is a short chain fatty acid, belongs to HDAC inhibitors which is released in the colonic lumen as a consequence of fiber fermentation. In this study, we are about to assess the effect of sodium butyrate on HCT116 human colorectal carcinoma cell line. The viability of cells was measured by microscopic morphologic study and MTT assay. After 48 hours, treatments more than 10 mM lead to cell injury in HCT116 by increasing cell granulation and decreasing cell adhesion (p>0.05). After 72 hours, treatments at 10 mM and more lead to significant cell injury (p<0.05). Our results may suggest that the gene expression which is contributed in cell proliferation and apoptosis has been changed under pressure of HDAC inhibition.

Keywords: colorectal cancer, sodium butyrate, cytotoxicity, MTT

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Authors: Bhumika Wadhwa, Fayaz Malik

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The serine/threonine protein kinase B (PKB) also known as Akt, is one of the multifaceted kinase in human kinome, existing in three isoforms. Akt plays a vital role in phosphoinositide 3-kinase (PI3K) mediated oncogenesis in various malignancies and is one of the attractive targets for cancer drug discovery. The functional significance of an individual isoform of Akt is not redundant in cancer cell proliferation and metastasis instead Akt isoforms play distinct roles during metastasis; thereby regulating EMT. This study aims to determine isoform specific functions of Akt in cancer. The results obtained suggest that Akt1 restrict tumor invasion, whereas Akt2 promotes cell migration and invasion by various techniques like MTT, wound healing and invasion assay. Similarly, qRT-PCR also revealed that Akt3 has shown promising results in promoting cancer cell migration. Contrary to pro-oncogenic properties attributed to Akt, it is to be understood how various isoforms of Akt compensates each other in the regulation of common pathways during cancer progression and drug resistance. In conclusion, this study aims to target selective isoforms which is essential to inhibit cancer. However, the question now is whether, and how much, Akt inhibition will be tolerated in the clinic remains to be answered and the experiments will have to address the question of which combinations of newly devised Akt isoform specific inhibitors exert a favourable therapeutic effect in in vivo models of cancer to provide the therapeutic window with minimal toxicity.

Keywords: Akt isoforms, cancer, drug resistance, epithelial mesenchymal transition

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Authors: Mahdieh Jajroudi

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Developing a pharmacokinetic model for the neuroendocrine tumors therapy agent 177Lu-DOTATATE in nude mice bearing AR42J rat pancreatic tumor to investigate and evaluate the behavior of the complex was the main purpose of this study. The utilization of compartmental analysis permits the mathematical differencing of tissues and organs to become acquainted with the concentration of activity in each fraction of interest. Biodistribution studies are onerous and troublesome to perform in humans, but such data can be obtained facilely in rodents. A physiologically based pharmacokinetic model for scaling up activity concentration in particular organs versus time was developed. The mathematical model exerts physiological parameters including organ volumes, blood flow rates, and vascular permabilities; the compartments (organs) are connected anatomically. This allows the use of scale-up techniques to forecast new complex distribution in humans' each organ. The concentration of the radiopharmaceutical in various organs was measured at different times. The temporal behavior of biodistribution of 177Lu labeled somatostatin analogues was modeled and drawn as function of time. Conclusion: The variation of pharmaceutical concentration in all organs is characterized with summation of six to nine exponential terms and it approximates our experimental data with precision better than 1%.

Keywords: biodistribution modeling, compartmental analysis, 177Lu labeled somatostatin analogues, neuroendocrine tumors

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Authors: Alexander A. Stakheev, Larisa V. Samokhvalova, Sergey K. Zavriev

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Fusarium fungi are among the most devastating plant pathogens distributed all over the world. Significant reduction of grain yield and quality caused by Fusarium leads to multi-billion dollar annual losses to the world agricultural production. These organisms can also cause infections in immunocompromised persons and produce the wide range of mycotoxins, such as trichothecenes, fumonisins, and zearalenone, which are hazardous to human and animal health. Identification of Fusarium fungi based on the morphology of spores and spore-forming structures, colony color and appearance on specific culture media is often very complicated due to the high similarity of these features for closely related species. Modern Fusarium taxonomy increasingly uses data of crossing experiments (biological species concept) and genetic polymorphism analysis (phylogenetic species concept). A number of novel Fusarium sibling species has been established using DNA barcoding techniques. Species recognition is best made with the combined phylogeny of intron-rich protein coding genes and ribosomal DNA sequences. However, the internal transcribed spacer of (ITS), which is considered to be universal DNA barcode for Fungi, is not suitable for genus Fusarium, because of its insufficient variability between closely related species and the presence of non-orthologous copies in the genome. Nowadays, the translation elongation factor 1 alpha (TEF1α) gene is the “gold standard” of Fusarium taxonomy, but the search for novel informative markers is still needed. In this study, we used two novel DNA markers, frataxin (FXN) and heat shock protein 90 (HSP90) to discover phylogenetic relationships between Fusarium species. Multilocus phylogenetic analysis based on partial sequences of TEF1α, FXN, HSP90, as well as intergenic spacer of ribosomal DNA (IGS), beta-tubulin (β-TUB) and phosphate permease (PHO) genes has been conducted for 120 isolates of 19 Fusarium species from different climatic zones of Russia and neighboring countries using maximum likelihood (ML) and maximum parsimony (MP) algorithms. Our analyses revealed that FXN and HSP90 genes could be considered as informative phylogenetic markers, suitable for evolutionary and taxonomic studies of Fusarium genus. It has been shown that PHO gene possesses more variable (22 %) and parsimony informative (19 %) characters than other markers, including TEF1α (12 % and 9 %, correspondingly) when used for elucidating phylogenetic relationships between F. avenaceum and its closest relatives – F. tricinctum, F. acuminatum, F. torulosum. Application of novel DNA barcodes confirmed the fact that F. arthrosporioides do not represent a separate species but only a subspecies of F. avenaceum. Phylogeny based on partial PHO and FXN sequences revealed the presence of separate cluster of four F. avenaceum strains which were closer to F. torulosum than to major F. avenaceum clade. The strain F-846 from Moldova, morphologically identified as F. poae, formed a separate lineage in all the constructed dendrograms, and could potentially be considered as a separate species, but more information is needed to confirm this conclusion. Variable sites in PHO sequences were used for the first-time development of specific qPCR-based diagnostic assays for F. acuminatum and F. torulosum. This work was supported by Russian Foundation for Basic Research (grant № 15-29-02527).

Keywords: DNA barcode, fusarium, identification, phylogenetics, taxonomy

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Authors: Abdul Qadir Qadis, Satoru Goya, Minoru Yatsu, Yu-uki Yoshida, Toshihiro Ichijo, Shigeru Sato

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Bacterial probiotics are known to modulate the gut-associated lymphoid and epithelial tissue response to enhance the activities of intestinal and systemic immune system in human and animals. In cattle, the immune-stimulatory effects of probiotics have been evaluated during intestinal disorders. To investigate the effects of probiotic on the function of peripheral blood mononuclear cells, eight healthy Holstein calves (10 ± 3 weeks) were assigned to a 4 × 2 experimental design. The probiotic, consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum, was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days. Calves given no probiotic served as the control. In the treatment group, increases in numbers of CD282+ monocytes, CD3+ T-cells and CD4+, CD8+ and WC1+ γδ T- cell subsets were noted on day 7 post-placement compared to pre-dose day and the control group. Expression of interleukin-6, interferon-gamma and tumor necrosis factor-alpha was elevated in peripheral leukocytes on days 7 and 14. These results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota, which was increased by the oral probiotic treatment. The 5-day repeated administration of a bacterial probiotic may enhance cellular immune function in weaned calves.

Keywords: bacterial-probiotic, calf, interleukin, leukocyte

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Authors: Mohamed A. Dkhil, Denis Delic, Saleh Al-Quraishy

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Coccidiosis causes considerable economic loss in the poultry industry. The current study aimed to investigate the response of goblet cells as well as the induced tissue damage during Eimeria papilata infection. Mice were infected with sporulated E. papillata oocyts. On day 5 post-infection, the fecal output was determined. Also, the jejunum was prepared for the histological, histochemical and molecular studies. Our results revealed that the intestinal coccidian infection with E. papillata induced a marked goblet cell hypoplasia and depleted mucus secretion. Also, the infection was able to alter the jejuna architecture and increased the apoptotic cells inside the villi. In addition, the real time PCR results indicated that, the inflammatory cytokines TNF-α, iNOS, IFN-y and IL-1β were significantly up-regulated. In contrast, the mRNA expression patterns of IL-6 in response to E. papillata infection did not differ significantly between control and infected mice. Moreover, the mRNA expression of TLR4 was significantly up-regulated, whereas the expression of MUC2 is significantly down-regulated upon infection. Further studies are required to understand the regulatory mechanisms of goblet cells related genes.

Keywords: goblet cells, Eimeria papillata, mice, jejunum

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Authors: Dominika Pihikova, Stefan Belicky, Tomas Bertok, Roman Sokol, Petra Kubanikova, Jan Tkac

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Since aberrant glycosylation is frequently accompanied by both physiological and pathological processes in a human body (cancer, AIDS, inflammatory diseases, etc.), the analysis of tumor-associated glycan patterns have a great potential for the development of novel diagnostic approaches. Moreover, altered glycoforms may assist as a suitable tool for the specificity and sensitivity enhancement in early-stage prostate cancer diagnosis. In this paper we discuss the construction and optimization of ultrasensitive sandwich biosensor platform employing lectin as glycan-binding protein. We focus on the immunoassay development, reduction of non-specific interactions and final glycoprofiling of human serum samples including both prostate cancer (PCa) patients and healthy controls. The fabricated biosensor was measured by label-free electrochemical impedance spectroscopy (EIS) with further lectin microarray verification. Furthermore, we analyzed different biosensor interfaces with atomic force microscopy (AFM) in nanomechanical mapping mode showing a significant differences in the altitude. These preliminary results revealing an elevated content of α-2,3 linked sialic acid in PCa patients comparing with healthy controls. All these experiments are important step towards development of point-of-care devices and discovery of novel glyco-biomarkers applicable in cancer diagnosis.

Keywords: biosensor, glycan, lectin, prostate cancer

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Authors: Jahanshah Ashkani, Kevin J. Naidoo

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Glycosylation is the major posttranslational modification (PTM) process in cellular development. In tumour development, it is marked by structural alteration of carbohydrates (glycans) that is the result of aberrant glycosylation. Altered glycan structures affect cell surface ligand-receptor interactions that interfere with the regulation of cell adhesion, migration, and proliferation. The resulting changes in glycan biosynthesis pathways originate from altered expression of glycosyltransferases and glycosidases. While the alteration in glycosylation patterns is a recognized “hallmark of cancer”, the influential overview of the biology of cancer proposes eight hallmarks with no explicit suggestion to connectivity with glycosylation. Recently, we have discovered a connection between the glycosyltransferase gene expression and cancer type and subtype. Here we present an association between aberrant glycosylation and the biological hallmarks of breast cancer by exploring the common regulatory mechanisms at the genomic scale. The result of this study bridges the glycobiological and biological pathways that are accepted hallmarks of cancer by connecting their common regulatory pathways. This is an impetus for further investigation as target therapies of breast cancer are very likely to be uncovered from this.

Keywords: aberrant glycosylation, biological hallmarks, breast cancer, regulatory mechanism

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Authors: Olga V. Chervyakova, Elmira T. Tailakova, Vitaliy M. Strochkov, Kulyaisan T. Sultankulova, Nurlan T. Sandybayev, Lev G. Nemchinov, Rosemarie W. Hammond

Abstract:

Sheep pox is a highly contagious infection that OIE regards to be one of the most dangerous animal diseases. It causes enormous economic losses because of death and slaughter of infected animals, lower productivity, cost of veterinary and sanitary as well as quarantine measures. To control spread of sheep pox infection the attenuated vaccines are widely used in the Republic of Kazakhstan and other Former Soviet Union countries. In spite of high efficiency of live vaccines, the possible presence of the residual virulence, potential genetic instability restricts their use in disease-free areas that leads to necessity to exploit new approaches in vaccine development involving recombinant DNA technology. Vaccines on the basis of recombinant proteins are the newest generation of prophylactic preparations. The main advantage of these vaccines is their low reactogenicity and this fact makes them widely used in medical and veterinary practice for vaccination of humans and farm animals. The objective of the study is to produce recombinant immunogenic proteins for development of the high-performance means for sheep pox prophylaxis. The SPV proteins were chosen for their homology with the known immunogenic vaccinia virus proteins. Assay of nucleotide and amino acid sequences of the target SPV protein genes. It has been shown that four proteins SPPV060 (ortholog L1), SPPV074 (ortholog H3), SPPV122 (ortholog A33) and SPPV141 (ortholog B5) possess transmembrane domains at N- or C-terminus while in amino acid sequences of SPPV095 (ortholog А 4) and SPPV117 (ortholog А 27) proteins these domains were absent. On the basis of these findings the primers were constructed. Target genes were amplified and subsequently cloned into the expression vector рЕТ26b(+) or рЕТ28b(+). Six constructions (pSPPV060ΔТМ, pSPPV074ΔТМ, pSPPV095, pSPPV117, pSPPV122ΔТМ and pSPPV141ΔТМ) were obtained for expression of the SPV genes under control of T7 promoter in Escherichia coli. To purify and detect recombinant proteins the amino acid sequences were modified by adding six histidine molecules at C-terminus. Induction of gene expression by IPTG was resulted in production of the proteins with molecular weights corresponding to the estimated values for SPPV060, SPPV074, SPPV095, SPPV117, SPPV122 and SPPV141, i.e. 22, 30, 20, 19, 17 and 22 kDa respectively. Optimal protocol of expression for each gene that ensures high yield of the recombinant protein was identified. Assay of cellular lysates by western blotting confirmed expression of the target proteins. Recombinant proteins bind specifically with antibodies to polyhistidine. Moreover all produced proteins are specifically recognized by the serum from experimentally SPV-infected sheep. The recombinant proteins SPPV060, SPPV074, SPPV117, SPPV122 and SPPV141 were also shown to induce formation of antibodies with virus-neutralizing activity. The results of the research will help to develop a new-generation high-performance means for specific sheep pox prophylaxis that is one of key moments in animal health protection. The research was conducted under the International project ISTC # K-1704 “Development of methods to construct recombinant prophylactic means for sheep pox with use of transgenic plants” and under the Grant Project RK MES G.2015/0115RK01983 "Recombinant vaccine for sheep pox prophylaxis".

Keywords: prophylactic preparation, recombinant protein, sheep pox virus, subunit vaccine

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Authors: Patience Orobosa Olajide

Abstract:

Five hydrocarbon degrading bacterial strains isolated from contaminated environment were investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Screening for bioplastic production was done on assay mineral salts agar medium containing 0.2% poly (3-hydroxybutyrate) as the sole carbon source. Two of the test bacteria were positive for PHB biosynthesis and were identified based on gram staining, biochemical tests, 16S rRNA gene sequence analysis as Pseudomonas aeruginosa and Bacillus licheniformis which grew at 37 and up to 65 °C respectively, thus suggesting the later to be thermotolerant. In this study, the effects of different carbon and nitrogen sources on PHB production in these strains were investigated. Maximum PHB production was obtained in 48 hr for the two strains and amounted to yields of 72.86 and 62.22 percentages for Bacillus licheniformis and Pseudomonas aeruginosa respectively. In these strains, glycine was the most efficient carbon sources for the production of PHB compared with other carbon (glucose, lactose, sucrose, Arabinose) and nitrogen (L- glycine, L-cysteine, DL-Tryptophan, and Potassium Nitrate) sources. The screening of microbial strains for industrial PHB production should be based on several factors including the cell’s capability to mineralize an inexpensive substrate, rate of growth and the extent of polymer accumulation.

Keywords: bacteria, poly-3-hydroxybutyrate (PHB), hydrocarbon, thermotolerant

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Authors: Shikha Masand, Sudesh Kumar Yadav

Abstract:

Certain protein kinases have been shown to be crucial for plant cell signaling pathways associated with plant immune responses. Here we identified a horsegram [Macrotyloma uniflorum (Lam.) Verdc.] malectin-like leucine rich receptor-like protein kinase (RLK) gene MuRLK. The functional MuRLK protein preferentially binds to mannose and N-acetyl glucosamine residues. MuRLK exists in the cytoplasm and also localizes to the plasma membrane of plant cells via its N-terminus. Over-expression of MuRLK in Arabidopsis enhances the basal resistance to infection with Pseudomonas syringae pv. tomato, Alternaria brassicicola and Hyaloperonospora arabidopsidis, are associated with elevated ROS bursts, MAPK activation, thus ultimately leading to hypersensitive cell death. Moreover, salicylic acid-dependent and jasmonic acid-dependent defense responses are also enhanced in the MuRLK-overexpressed plants that lead to HR-induced cell death. Together, these results suggest that MuRLK plays a key role in the regulation of plant cell death, early and late defense responses after the recognition of microbial pathogens.

Keywords: horsegram, Pseudomonas syringae pv. tomato, MuRLK, ROS burst, cell death, plant defense

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Authors: Piamsiri Sawaisorn, Tienrat Tangchaikeeree, Waraporn Chan-On, Chaniya Leepiyasakulchai, Rachanee Udomsangpetch, Suradej Hongeng, Kulachart Jangpatarapongsa

Abstract:

Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition and non-major histocompatibility complex (MHC) restriction. An issue of recent interest is the capability to activate γδ T cells by use of a group of drugs, such as pamidronate, that cause accumulation of phosphoantigen which is recognized by γδ T cell receptors. Moreover, their antigen presenting cell-like phenotype and function have been confirmed in many clinical trials. In this study, Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with pamidronate and the expanded Vγ9Vδ2 T cells can recognize and kill chronic myeloid leukemia (CML) cells treated with pamidronate through their cytotoxic activity. To support the strong role played by Vγ9Vδ2 T cells against cancer, we provide the evidence that Vγ9Vδ2 T cells activated with CML cell lysate antigen can efficiently express antigen presenting cell (APC) phenotype and function. In conclusion, pamidronate can be used in intentional activation of human Vγ9Vδ2 T cells and can increase the susceptibility of CML cells to cytotoxicity of Vγ9Vδ2 T cells. The activated Vγ9Vδ2 T cells by cancer cells lysate can show their APC characteristics, and so greatly increase the interest in exploring their therapeutic potential in hematologic malignancy.

Keywords: γδ T lymphocytes, antigen-presenting cells, chronic myeloid leukemia, cancer, immunotherapy

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Authors: Darshan Panda, Lambodar Behera, M. J. Baig, Sudhanshu Sekhar

Abstract:

Low light intensity is a significant limitation for grain yield and quality in rice. However, yield is not significantly reduced in low-light tolerant rice varieties. The work, therefore, planned for comparative transcriptome profiling under low light stress to decipher the genes involved and molecular mechanism of low light tolerance in rice. At the active tillering stage, 50% low light exposure for one day, three days, and five days were given to Swarnaprabha (low light tolerant) and IR8 (low light sensitive) rice varieties. Illumina (HiSeq) platform was used for transcriptome sequencing. A total of 6,652 and 12,042 genes were differentially expressed due to low light intensity in Swarnaprabha and IR8, respectively, as compared to control. CAB, LRP, SBPase, MT15, TF PCL1, and Photosystem I & II complex related gene expressions were mostly increased in Swarnaprabha upon the longer duration of low light exposure, which was not found in IR8 as compared to control. Their expressions were validated by qRT-PCR. The overall study suggested that the maintenance of grain yield in the tolerant variety under low light might be the result of accelerated expression of the genes, which enable the plant to keep the photosynthetic processes moving at the same pace even under low light.

Keywords: rice, low light, photosynthesis, yield

Procedia PDF Downloads 194

Authors: Michael Josef Schwerer

Abstract:

Background: Analyzing any aircraft accident is mandatory based on the regulations of the International Civil Aviation Organization and the respective country’s criminal prosecution authorities. Legal medicine investigations are unavoidable when fatalities involve the flight crew or when doubts arise concerning the pilot’s aeromedical health status before the event. As a result of frequently tremendous blunt and sharp force trauma along with the impact of the aircraft to the ground, consecutive blast or fire exposition of the occupants or putrefaction of the dead bodies in cases of delayed recovery, relevant findings can be masked or destroyed and therefor being inaccessible in standard pathology practice comprising just forensic autopsy and histopathology. Such cases are of considerable risk of remaining unsolved without legal consequences for those responsible. Further, no lessons can be drawn from these scenarios to improve flight safety and prevent future mishaps. Aims and Methods: To learn from previously unsolved aircraft accidents, re-evaluations of the investigation files and modern molecular pathology studies were performed. Genetic testing involved predominantly PCR-based analysis of gene regulation, studying DNA promotor methylations, RNA transcription and posttranscriptional regulation. In addition, the presence or absence of infective agents, particularly DNA- and RNA-viruses, was studied. Technical adjustments of molecular genetic procedures when working with archived sample material were necessary. Standards for the proper interpretation of the respective findings had to be settled. Results and Discussion: Additional molecular genetic testing significantly contributes to the quality of forensic pathology assessment in aviation mishaps. Previously undetected cardiotropic viruses potentially explain e.g., a pilot’s sudden incapacitation resulting from cardiac failure or myocardial arrhythmia. In contrast, negative results for infective agents participate in ruling out concerns about an accident pilot’s fitness to fly and the aeromedical examiner’s precedent decision to issue him or her an aeromedical certificate. Care must be taken in the interpretation of genetic testing for pre-existing diseases such as hypertrophic cardiomyopathy or ischemic heart disease. Molecular markers such as mRNAs or miRNAs, which can establish these diagnoses in clinical patients, might be misleading in-flight crew members because of adaptive changes in their tissues resulting from repeated mild hypoxia during flight, for instance. Military pilots especially demonstrate significant physiological adjustments to their somatic burdens in flight, such as cardiocirculatory stress and air combat maneuvers. Their non-pathogenic alterations in gene regulation and expression will likely be misinterpreted for genuine disease by inexperienced investigators. Conclusions: The growing influence of molecular pathology on legal medicine practice has found its way into aircraft accident investigation. As appropriate quality standards for laboratory work and data interpretation are provided, forensic genetic testing supports the medico-legal analysis of aviation mishaps and potentially reduces the number of unsolved events in the future.

Keywords: aviation medicine, aircraft accident investigation, forensic pathology, molecular pathology

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Authors: Dalia O. Saleha, Gehad A. Abdel Jaleela, Sally W. Al-Awdana

Abstract:

Diabetes mellitus (DM) is known to exacerbate cerebral ischemic injury. The present study aimed to investigate the anti-oxidant and anti-inflammatory effects of oral supplementation of melatonin (MLN) on cerebral injury caused by middle cerebral artery occlusion and reperfusion (MCAO/Re) in streptozotocin (STZ)-induced hyperglycemic rats. Hyperglycemia was induced by a single injection of STZ (55mg/kg; i.p.), six weeks later the cerebral injury was induced by MCAO/Re. Twenty-four hours after the MCAO/Re the MLN (10 mg/kg) was injected for 14 consecutive days. Results of the present study revealed that MCAO/Re in STZ-induced hyperglycemia in rats causes an increase in the oxidative stress biomarkers; it increased brain lipid peroxidation (measured as malondialdehyde; MDA) and brain level of nitric oxide (NO). Moreover, MCAO/Reproduces a prominent increase in the brain inflammatory markers viz. interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis nuclear factor-alpha (TNF-α). Oral treatment of MCAO/Re in STZ-induced hyperglycemic rats with MLN (10 mg/kg) for two weeks restored the brain levels of MDA, GSH, NO, IL-6, IL-1β and the TNF-α. MLN succeeded to suppress the exacerbation of damage in the brain of hyperglycemic rats. These results suggest that daily intake of MLN attenuates the exacerbation of cerebral ischemic injury in a diabetic state, which may be attributed to anti-oxidant and anti-inflammatory effects in the brain.

Keywords: melatonin, brain injury, cerebral ischemia/reperfusion, hyperglycemia, rats

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Authors: Deepa Gandhi, Pravin K. Naoghare, Amit Bafana, Krishnamurthi Kannan, Saravanadevi Sivanesana

Abstract:

Oxidative stress is known to depreciate normal functioning of osteoblast cells. Present study reports oxidative/inflammatory signatures in fluoride exposed human osteosarcoma (HOS) cells and its possible association with the genes involved in bone developmental pathway. Microarray analysis was performed to understand the possible molecular mechanisms of stress-mediated bone lose in HOS cells. Cells were chronically exposed with sub-lethal concentration of fluoride. Global gene expression is profiling revealed 34 up regulated and 2598 down-regulated genes, which were associated with several biological processes including bone development, osteoblast differentiation, stress response, inflammatory response, apoptosis, regulation of cell proliferation. Microarray data were further validated through qRT-PCR and western blot analyses using key representative genes. Based on these findings, it can be proposed that chronic exposure of fluoride may impair bone development via oxidative and inflammatory stress. The present finding also provides important biological clues, which will be helpful for the development of therapeutic targets against diseases related bone.

Keywords: bone, HOS cells, microarray, stress

Procedia PDF Downloads 377

Authors: Hsin-Lien Lin, Ju-Hui Fu

Abstract:

Dendritic cells (DCs) are the major professional antigen-presenting cells for the development of optimal T-cell immunity. DCs can be used as pharmacological targets to screen novel biological modifiers for the treatment of harmful immune responses, such as transplantation rejection. Dryopteris crassirhizoma Nakai (Aspiadaceae) is used for traditional herbal medicine in the region of East Asia. The root of this fern plant has been listed for treating inflammatory diseases. Dryocrassin is the tetrameric phlorophenone component derived from Dryopteris. Here, we tested the immunomodulatory potential of dryocrassin on lipopolysaccharide (LPS)-stimulated activation of mouse bone marrow-derived DCs in vitro and in skin allograft transplantation in vivo. Results demonstrated that dryocrassin reduced the secretion of tumor necrosis factor-α, interleukin-6, and interleukin-12p70 by LPS-stimulated DCs. The expression of LPS-induced major histocompatibility complex class II, CD40, and CD86 on DCs was also blocked by dryocrassin. Moreover, LPS-stimulated DC-elicited allogeneic T-cell proliferation was lessened by dryocrassin. In addition, dryocrassin inhibited LPS-induced activation of IϰB kinase, JNK/p38 mitogen-activated protein kinase, as well as the translocation of NF-ϰB. Treatment with dryocrassin obviously diminished 2,4-dinitro-1-fluorobenzene- induced delayed-type hypersensitivity and prolonged skin allograft survival. Dryocrassin may be one of the potent immunosuppressive agents for transplant rejection through the destruction of DC maturation and function.

Keywords: dryocrassin, dendritic cells, immunosuppression, skin allograft

Procedia PDF Downloads 386

Authors: Mai M. Farid, Ximeng Yang, Tomoharu Kuboyama, Yuna Inada, Chihiro Tohda

Abstract:

Intro: Trigonella foenum-graecum (Fenugreek), from Fabaceae family is a well-known plant traditionally used as food and medicine. Many pharmacological effects of Trigonella foenum- graecum seeds extract (TF extract) were evaluated such as anti-diabetic, anti-tumor and anti-dementia effects using in vivo models. Regarding the anti-dementia effects of TF extract, diabetic rats, aluminum chloride-induced amnesia rats and scopolamine-injected mice were used previously for evaluation, which are not well established as Alzheimer’s disease models. In addition, those previous studies, active constituents in TF extract for memory function were not identified. Method: This study aimed to clarify the effect of TF extract on Alzheimer’s disease model, 5XFAD mouse that overexpresses mutated APP and PS1 genes and determine the major active constituent in the brain after oral intake of TF extract. Results: Trigonelline was detected in the cerebral cortex of 5XFAD mice after 24 hours of oral administration of TF extract by LC-MS/MS. Oral administration of TF extract for 17 days improved object location memory in 5XFAD mice. Conclusion: These results suggest that TF extract and its active constituents could be an expected therapeutic candidate for Alzheimer’s disease.

Keywords: Alzheimer's disease, LC-MS/MS, memory recovery, Trigonella foenum-graecum Seeds, 5XFAD mice

Procedia PDF Downloads 147

Authors: Safa Abusara Mohammed Ali, Mohammed Khair Abdallah, Gurdon A. Brockmann, M. Reissmann

Abstract:

The aim of the study was the characterization of DGAT1 variants in Sudanese dairy cattle breeds. In this study, we examined 94 Kenana and 91 Butana dairy cattle from two regions of Sudan. We genotyped the DGAT1 sequence variant AJ318490.1:g.10433/10434 AA>GC that leads to the Lysine – Alanine substitution at position 232 (K232A) in the protein and the VNTR polymorphism in the promoter region. Genotyping was performed by allele specific PCR and PCR fragment lengths determination, respectively. In both breeds, the DGAT1 Lysine variant (232K) that is associated with high fat and protein content as well as high fat yield in other breeds is the high frequent allele. The frequencies of the 232K allele were 96.3% and 84.6% in Kenana and Butana breeds, respectively. At the DGAT1 promoter VNTR locus, four alleles containing four to seven repeats of the 18 bp motif were found in both breeds. The highest frequent allele was the VNTR allele 3 containing five repeats with 60.4 % and 57.5 % in Kenana and Butana breeds, respectively. In conclusion, the two examined Sudanese dairy cattle breeds do not differ in allele frequencies at the DGAT1 locus.

Keywords: dairy cattle, DGAT1, Kenana, Butana.

Procedia PDF Downloads 121