Search results for: quantitative mRNA expression

Authors: Mohd Ali Abbas Zaidi, Meenu Sachdeva, Jonaki Sen

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In the vertebrate embryo, the forebrain anlagen develops from the anterior-most region of the neural tube which is the precursor of the central nervous system (CNS). The roof plate located at the dorsal midline region of the forebrain anlagen, acts as a source of several secreted molecules involved in patterning and morphogenesis of the forebrain. One such key morphogenetic event is the invagination of the forebrain roof plate which results in separation of the single forebrain vesicle into two cerebral hemispheres. Retinoic acid (RA) signaling plays a key role in this process. Blocking RA signaling at the dorsal forebrain midline inhibits dorsal invagination and results in the absence of certain key features of this region, such as thinning of the neuroepithelium and a lowering of cell proliferation. At present we are investigating the possibility of other signaling pathways acting in concert with RA signaling to regulate this process. We have focused on BMP signaling, which we found to be active in a mutually exclusive domain to that of RA signaling within the roof plate. We have also observed that there is a change in BMP signaling activity on modulation of RA signaling indicating an antagonistic relationship between the two. Moreover, constitutive activation of BMP signaling seems to completely inhibit thinning and partially affect invagination, leaving the lowering of cell proliferation in the midline unaffected. We are employing in-silico modeling as well as molecular manipulations to investigate the relative contribution if any, of regional differences in rates of cell proliferation and thinning of the neuroepithelium towards the process of invagination. We have found expression of certain cell adhesion molecules in forebrain roof-plate whose mRNA localization across the thickness of neuroepithelium is influenced by Bmp and RA signaling, giving regional rigidity to roof plate and assisting invagination. We also found expression of certain cytoskeleton modifiers in a localized small domains in invaginating forebrain roof plate suggesting that midline invagination is under control of many factors.

Keywords: bone morphogenetic signaling, cytoskeleton, cell adhesion molecules, forebrain roof plate, retinoic acid signaling

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Authors: Mariam G. Eshak, Ahmed Abbas, M. I. El-Sabry, M. M. Mashaly

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This investigation was conducted to determine the physiological response and evaluate the expression of inflammatory and cell death genes and DNA damage induced by endotoxic shock in laying hens. Endotoxic shock was induced by a single intravenous injection of 107 Escherichia coli (E. coli,) colony/hen. In the present study, 240 forty-week-old laying hens (H&N) were randomly assigned into 2 groups with 3 replicates of 40 birds each. Hens were reared in battery cages with wire floors in an open-sided housing system under natural conditions. Housing and general management practices were similar for all groups. At 42-wk of age, 45 hens from the first group (15 replicate) were infected with E. coli, while the same number of hens from the second group was injected with saline and served as a control. Heat shock protein-70 (HSP-70) expression, plasma corticosterone concentration, body temperature, and the gene expression of bax, caspase-3 activity, P38, Interlukin-1β (Il-1β), and tumor necrosis factor alpha (TNF-α) genes and DNA damage in the brain and liver were measured. Hens treated with E. coli showed significant (P≤0.05) increase of body temperature by 1.2 ᴼC and plasma corticosterone by 3 folds compared to the controls. Further, hens injected with E.Coli showed markedly over-expression of HSP-70 and increase DNA damage in brain and liver. These results were synchronized with activating cell death program since our data showed significant (P≤0.05) high expression of bax and caspase-3 activity genes in the brain and liver. These results were related to remarkable over-inflammation gene expression of P38, IL-1β, and TNF-α in brain and liver. In conclusion, our results indicate that endotoxic shock induces inflammatory physiological response and triggers cell death program by promoting P38, IL-1β, and TNF-α gene expression in the brain and liver.

Keywords: chicken, DNA damage, Escherichia coli, gene expression, inflammation

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Authors: Doaa Hashad, Amany Elbanna, Abeer Ibrahim, Gihan Khedr

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Background: H19 is one of the long non coding RNAs (LncRNA) that is related to the progression of many diseases including cancers. This work was carried out to study the level of the long non-coding RNA; H119, in plasma of patients with gastric cancer (GC) and to assess its significance in their clinical management. Methods: A total of sixty-two participants were enrolled in the present study. The first group included thirty-two GC patients, while the second group was formed of thirty age and sex matched healthy volunteers serving as a control group. Plasma samples were used to assess H19 gene expression using real time quantitative PCR technique. Results: H19 expression was up-regulated in GC patients with positive correlation to TNM cancer stages. Conclusions: Up-regulation of H19 is closely associated with gastric cancer and correlates well with tumor staging. Convenient, efficient quantification of H19 in plasma using real time PCR technique implements its role as a potential noninvasive prognostic biomarker in gastric cancer, that predicts patient’s outcome and most importantly as a novel target in gastric cancer treatment with better performance achieved on using both CEA and H19 simultaneously.

Keywords: biomarker, gastric, cancer, LncRNA

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Authors: Chun Hsien Wu, Guan Xuan Wu, Hsia Ying Cheng, Shyh Ming Kuo

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Articular cartilage is composed of chondrocytes and extracellular matrix, such as collagen fibers, glycosaminoglycans, etc., which play an important role in lubricating and cushion joint activities. The phenotypic expression and metabolic activity of chondrocytes are extremely important in maintaining the functions of articular cartilage. In in vitro passaged culture of chondrocytes, chondrocytes gradually lose their original cell phenotype and morphology, which is called dedifferentiation. After continuous passaged culture of chondrocytes or induction by inflammatory factor IL-1, chondrocytes changed their phenotype and morphology. Also, the extracellular matrix type II collagen and GAG secretion were significantly reduced, while type I and X collagen were synthesized. Farnesol is an anti-inflammatory and antioxidant sesquiterpene compound that has the specific property of promoting collagen production. The purpose of this study was to investigate whether farnesol could restore the original type II collagen synthesis and, furthermore, the mechanisms of farnesol on the synthesis of type II collagen from the de-differentiated chondrocytes. The obtained results showed that the de-differentiated chondrocytes significantly restored to secret type II collagen and GAG (2.5-folds increases), and the secretion of collagen I and X and PGE2 synthesis were also significantly reduced after being treated with farnesol, indicating that farnesol had a restoration/re-differentiation effect on de-differentiated chondrocytes. The de-differentiated chondrocytes exhibited decreased expression of PPAR-γ and upregulated TGF-β expression to increase the MMP-13 expression. Higher expression of MMP-13 caused chondrocytes to secret type X collagen. On the contrary, increasing the expression of PPAR-γ would benefit the production of type II collagen. As shown, the PPAR-γ expression increased, and MMP-13 expression decreased after being treated with farnesol, indicating a possible signal pathway of farnesol to restore the production of type II collagen. However, more detailed mechanisms still need to evaluate.

Keywords: chondrocytes, de-differentiation, farnesol, re-differentiation

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Authors: Shohei Maruyama, Yasuo Matsuyama, Sachiyo Aburatani

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The development of the method to annotate unknown gene functions is an important task in bioinformatics. One of the approaches for the annotation is The identification of the metabolic pathway that genes are involved in. Gene expression data have been utilized for the identification, since gene expression data reflect various intracellular phenomena. However, it has been difficult to estimate the gene function with high accuracy. It is considered that the low accuracy of the estimation is caused by the difficulty of accurately measuring a gene expression. Even though they are measured under the same condition, the gene expressions will vary usually. In this study, we proposed a feature extraction method focusing on the variability of gene expressions to estimate the genes' metabolic pathway accurately. First, we estimated the distribution of each gene expression from replicate data. Next, we calculated the similarity between all gene pairs by KL divergence, which is a method for calculating the similarity between distributions. Finally, we utilized the similarity vectors as feature vectors and trained the multiclass SVM for identifying the genes' metabolic pathway. To evaluate our developed method, we applied the method to budding yeast and trained the multiclass SVM for identifying the seven metabolic pathways. As a result, the accuracy that calculated by our developed method was higher than the one that calculated from the raw gene expression data. Thus, our developed method combined with KL divergence is useful for identifying the genes' metabolic pathway.

Keywords: metabolic pathways, gene expression data, microarray, Kullback–Leibler divergence, KL divergence, support vector machines, SVM, machine learning

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Authors: Alexander V. Spirov, David M. Holloway, Ekaterina M. Myasnikova

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Segmentation of the early Drosophila embryo results from the dynamic establishment of spatial gene expression patterns. Patterning occurs on an embryo geometry which is a 'deformed' prolate ellipsoid, with anteroposterior and dorsal-ventral major and minor axes, respectively. Patterning is largely independent along each axis, but some interaction can be seen in the 'bending' of the segmental expression stripes. This interaction is not well understood. In this report, we investigate how 3D geometrical features of the early embryo affect the segmental expression patterning. Specifically, we study the effect of geometry on formation of the Bicoid primary morphogenetic gradient. Our computational results demonstrate that embryos with a much longer ventral than dorsal surface ('bellied') can produce curved Bicoid concentration contours which could activate curved stripes in the downstream pair-rule segmentation genes. In addition, we show that having an extended source for Bicoid in the anterior of the embryo may be necessary for producing the observed exponential form of the Bicoid gradient along the anteroposterior axis.

Keywords: Drosophila embryo, bicoid morphogenetic gradient, exponential expression profile, expression surface form, segmentation genes, 3D modelling

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Authors: Abdullah M. Alzahrani

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Our physiopathological assumption is that u-PA, t-PA, and PAI-1 are released by calcified aortic valves and play a role in the calcification of these valves. Sixty-five calcified aortic valves were collected from patients suffering from aortic stenosis. Each valve was incubated for 24 hours in culture medium. The supernatants were used to measure u-PA, t-PA, and PAI-1 concentrations; the valve calcification was evaluated using biphotonic absorptiometry. Aortic stenosis valves expressed normal plasminogen activators concentrations and overexpressed PAI-1 (u-PA, t-PA, and PAI-1 mean concentrations were, resp., 1.69 ng/mL ± 0.80, 2.76 ng/mL ± 1.33, and 53.27 ng/mL ± 36.39). There was no correlation between u-PA and PAI-1 (r = 0.3) but t-PA and PAI-1 were strongly correlated with each other (r = 0.6). Over expression of PAI-1 was proportional to the calcium content of theAS valves. Our results demonstrate a consistent increase of PAI-1 proportional to the calcification. The over expression of PAI-1 may be useful as a predictive indicator in patients with aortic stenosis.

Keywords: aortic valve, PAI-1, tPA gene, uPA gene

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Authors: Li Yuchen, Wu Qingxin, Jin Yuxing, Yang Qian

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Interleukin-11 (IL-11), a well-known anti-inflammatory factor, helps to protect against intestinal epithelium damage caused by physical or chemical factors. However, little is known about the role of IL-11 during viral infection. Herein, high mRNA and protein levels of IL-11 were found in epithelial cells and jejunum of piglets during porcine epidemic diarrhea virus (PEDV) infection, and IL-11 expression was positively correlated with the level of viral infection. Pretreatment with recombinant porcine IL-11 (pIL-11) suppressed PEDV replication in Vero E6 cells, while IL-11 knockdown promoted viral infection. Furthermore, pIL-11 inhibited viral infection by preventing PEDV-mediated apoptosis of cells through activating the IL-11/STAT3 signal pathway. Conversely, application of a STAT3 phosphorylation inhibitor significantly antagonized the anti-apoptosis function of pIL-11 and counteracted its inhibition of PEDV. Our data suggested that that IL-11 is a novel PEDV-inducible cytokine, and its production enhances the anti-apoptosis ability of epithelial cells against PEDV infection. The potential uses of IL-11 as a novel therapeutic against devastating viral diarrhea in piglets deserves more attention and study.

Keywords: Interleukin-11, Porcine epidemic diarrhea virus, STAT3, anti-apoptosis

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Authors: Kamila Weissova, Jitka Skrabalova, Katerina Skalova, Jana Koprivova, Zdenka Bendova

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Any Arctic visitor has to deal with extreme conditions, including constant light during the summer season or constant darkness during winter time. Light/dark cycle is the most powerful synchronizing signal for biological clock and the absence of daily dark period during the polar day can significantly alter the functional state of the internal clock. However, the inner clock can be synchronized by other zeitgebers such as physical activity, food intake or social interactions. Here, we investigated the effect of polar day on circadian clock of 10 researchers attending the polar base station in the Svalbard region during July. The data obtained on Svalbard were compared with the data obtained before the researchers left for the expedition (in the Czech Republic). To determine the state of circadian clock we used wrist actigraphy followed by sleep diaries, saliva, and buccal mucosa samples, both collected every 4 hours during 24h-interval to detect melatonin by radioimmunoassay and clock gene (PER1, BMAL1, NR1D1, DBP) mRNA levels by RT-qPCR. The clock gene expression was analyzed using cosinor analysis. From our results, it is apparent that the constant sunlight delayed melatonin onset and postponed the physical activity in the same order. Nevertheless, the clock gene expression displayed higher amplitude on Svalbard compared to the amplitude detected in the Czech Republic. These results have suggested that the common daily schedule at the Svalbard expedition can strengthen circadian rhythm in the environment that is lacking light/dark cycle. In conclusion, the constant sunlight delays melatonin onset, but it still maintains its rhythmic secretion. The effect of constant sunlight on circadian clock can be minimalized by common daily scheduled activity.

Keywords: actighraph, clock genes, human, melatonin, polar day

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Authors: V. Yurina, H. Sujuti, E. Rahmani, A. R. Nopitasari

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Breast cancer has the highest prevalence cancer in women. Moringa leaves (M. oleifera) contain quercetin, kaempferol, and benzyl isothiocyanate which can enhance induction of apoptosis. This research aimed to study the role of the leaf extract of Moringa to increase apoptosis in breast cancer cell line, MCF-7 cells. This research used in vitro experimental, post-test only, control group design on breast cancer cells MCF-7 in vitro. Moringa leaves were extracted by maceration method with ethanol 70%. Cells were treated with drumstick leaves extract on 1100, 2200, and 4400 μg/ml for Hsp27 and caspase-9 expression (immunocytochemistry) and apoptosis (TUNEL assay) test. The results of this study found that the IC50 2200 µg/ml. Moringa leaves extract can significantly increase the expression of caspase-9 (p<0.05) and decreased Hsp 27 expression (p<0.05). Moreover it can increase apoptosis (p<0.05) significantly in MCF-7 cells. The conclusion of this study is Moringa leaves extract is able to increase the expression of caspase-9, decrease Hsp27 expression and increase apoptosis in breast cancer cell-line MCF-7.

Keywords: apoptosis, breast cancer, caspase-9, Hsp27, Moringa oleifera

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Authors: Pattanapon Kayansamruaj, Ha Thanh Dong, Nopadon Pirarat, Channarong Rodkhum

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Streptococcus iniae (SI) is a devastating pathogenic bacteria causing heavy mortality in farmed fish. The application of commercialized bacterin vaccine has been reported failures as the outbreaks of the new serotype of SI were emerged in farms after vaccination and subsequently caused severe losses. In the present study, we attempted to develop effective DNA vaccines against SI infection using Nile tilapia (Oreochromis niloticus) as an animal model. Two monovalent DNA vaccines were constructed by the insertion of coding sequences of cell wall-associated virulence factors-encoding genes, comprised of eno (α-enolase) and mtsB (hydrophobic membrane protein), into cytomegalovirus expression vector (pCI-neo). In the animal trial, 30-g Nile tilapia were injected intramuscularly with 15 µg of each vaccine (mock vaccine group was injected by naked pCI-neo) and maintained for 35 days prior challenging with pathogenic SI at the dosage of 107 CFU/fish. At 13 days post-challenge, the relative percent survival of pEno, pMtsB and mock vaccine were 57%, 45% and 27%, respectively. The expression levels of immune responses-associated genes, namely, IL1β, TNF-α, TGF-β, COX2, IL-6, IL-12 and IL-13, were investigated from the spleen of experimental animal at 7 days post-vaccination (PV) and 7 days post-challenge (PC) using quantitative RT-PCR technique. Generally, at 7 days PV, the pEno vaccinated group exhibited highest level of up-regulation (1.7 to 2.9 folds) of every gene, but TGF-β, comparing to pMtsB and mock vaccine groups. However, at 7 days PC, pEno group showed significant up-regulation (1.4 to 8.5 folds) of immune-related genes as similar as mock vaccine group, while pMtsB group had lowest level of up-regulation (0.7 to 3.3 folds). Summarily, this study indicated that the pEno and pMtsB vaccines could elicit the immune responses of the fish and the magnitude of gene expression at 7 days PV was also consistent with the protection level conferred by the vaccine.

Keywords: gene expression, DNA vaccine, Nile tilapia, Streptococcus iniae

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Authors: Wan-Chun Chang, Yi-Jung Lee

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Due to cultural factors, expressing emotions may not be encouraged in collectivist cultures, which emphasize the needs of the group over the needs of the individual. This phenomenon is more prominent for children of migratory families. Due to the absence of one parent, children were often parentified by adults, which then impacted on their self-differentiation process. It made them more difficult to express their needs and emotions freely and openly. This study aimed to investigate the meditation effect of self-differentiation between parentification, and ambivalence over emotional expression for children of migratory families in Taiwan. Participants included 460 (326 females, 134 males) Taiwanese adults (age 18-25 years). The data were collected through questionnaires and analyzed using descriptive statistics and multiple regression analysis. The questionnaire included informed consent form, 'Filial Responsibility Scale-Adult', 'Chinese version of the Differentiation of Self Inventory', 'Ambivalence over Emotion Expressiveness Questionnaire', and the demographic sheet. Results indicated that self-differentiation mediated the relationship between parentified experience and ambivalence over emotional expression. In other words, parentified experience itself does not have the power to affect ambivalence over emotional expression. Only by affecting self-differentiation can it make an actual difference. The results were as expected and confirmed the hypothesis. Implications for clinical practice, research, and training were discussed.

Keywords: ambivalence over emotional expression, children of migratory families, parentification, self-differentiation

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Authors: Ankan Roy, Niharika, Samir Kumar Patra

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Anomalous nexus of complex topological assemblies and spatiotemporal epigenetic choreography at chromosomal territory may forms the most sophisticated regulatory layer of gene expression in cancer. Colon cancer is one of the leading malignant neoplasms of the lower gastrointestinal tract worldwide. There is still a paucity of information about the complex molecular mechanisms of colonic cancerogenesis. Bioinformatics prediction and analysis helps to identify essential genes and significant pathways for monitoring and conquering this deadly disease. The present study investigates and explores potential hub genes as biomarkers and effective therapeutic targets for colon cancer treatment. Colon cancer patient sample containing gene expression profile datasets, such as GSE44076, GSE20916, and GSE37364 were downloaded from Gene Expression Omnibus (GEO) database and thoroughly screened using the GEO2R tool and Funrich software to find out common 2 differentially expressed genes (DEGs). Other approaches, including Gene Ontology (GO) and KEGG pathway analysis, Protein-Protein Interaction (PPI) network construction and hub gene investigation, Overall Survival (OS) analysis, gene correlation analysis, methylation pattern analysis, and hub gene-Transcription factors regulatory network construction, were performed and validated using various bioinformatics tool. Initially, we identified 166 DEGs, including 68 up-regulated and 98 down-regulated genes. Up-regulated genes are mainly associated with the Cytokine-cytokine receptor interaction, IL17 signaling pathway, ECM-receptor interaction, Focal adhesion and PI3K-Akt pathway. Downregulated genes are enriched in metabolic pathways, retinol metabolism, Steroid hormone biosynthesis, and bile secretion. From the protein-protein interaction network, thirty hub genes with high connectivity are selected using the MCODE and cytoHubba plugin. Survival analysis, expression validation, correlation analysis, and methylation pattern analysis were further verified using TCGA data. Finally, we predicted COL1A1, COL1A2, COL4A1, SPP1, SPARC, and THBS2 as potential master regulators in colonic cancerogenesis. Moreover, our experimental data highlights that disruption of lipid raft and RAS/MAPK signaling cascade affects this gene hub at mRNA level. We identified COL1A1, COL1A2, COL4A1, SPP1, SPARC, and THBS2 as determinant hub genes in colon cancer progression. They can be considered as biomarkers for diagnosis and promising therapeutic targets in colon cancer treatment. Additionally, our experimental data advertise that signaling pathway act as connecting link between membrane hub and gene hub.

Keywords: hub genes, colon cancer, DNA methylation, epigenetic engineering, bioinformatic predictions

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Authors: Sadaf Ilyas

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Recombinant cytokines have been employed successfully as potential therapeutic agent. Some cytokine therapies are already used as a part of clinical practice, ranging from early exploratory trials to well established therapies that have already received approval. Interleukin 15 is a pleiotropic cytokine having multiple roles in peripheral innate and adaptive immune cell function. It regulates the activation, proliferation and maturation of NK cells, T-cells, monocytes/macrophages and granulocytes, and the interactions between them thus acting as a bridge between innate and adaptive immune responses. Unraveling the biology of IL-15 has revealed some interesting surprises that may point toward some of the first therapeutic applications for this cytokine. In this study, the human interleukin 15 gene was isolated, amplified and ligated to a TA vector which was then transfected to a bacterial host, E. coli Top10F’. The sequence of cloned gene was confirmed and it showed 100% homology with the reported sequence. The confirmed gene was then subcloned in pET Expression system to study the IPTG induced expression of IL-15 gene. Positive expression was obtained for number of clones that showed 15 kd band of IL-15 in SDS-PAGE analysis, indicating the successful strain development that can be studied further to assess the potential therapeutic intervention of this cytokine in relevance to human diseases.

Keywords: Interleukin 15, pET expression system, immune therapy, protein purification

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Authors: Ekaterina M. Myasnikova, Andrey A. Makashov, Alexander V. Spirov

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First manifestation of a segmentation pattern in the early Drosophila development is the formation of expression domains (along with the main embryo axis) of genes belonging to the trunk gene class. Highly variable expression of genes from gap family in early Drosophila embryo is strongly reduced by the start of gastrulation due to the gene cross-regulation. The dynamics of gene expression is described by a gene circuit model for a system of four gap genes. It is shown that for the formation of a steep and stationary border by the model it is necessary that there existed a nucleus (modeling point) in which the gene expression level is constant in time and hence is described by a stationary equation. All the rest genes expressed in this nucleus are in a dynamic equilibrium. The mechanism of border formation associated with the existence of a stationary nucleus is also confirmed by the experiment. An important advantage of this approach is that properties of the system in a stationary nucleus are described by algebraic equations and can be easily handled analytically. Thus we explicitly characterize the cross-regulation properties necessary for the robustness and formulate the conditions providing this effect through the properties of the initial input data. It is shown that our formally derived conditions are satisfied for the previously published model solutions.

Keywords: drosophila, gap genes, reaction-diffusion model, robustness

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Authors: Rasha Ahmadi

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

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

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Authors: Abhaydeep Pandey, Shweta Shukla, Saptamita Goswami, Bhaswati Bandyopadhyay, Vishnampettai Ramachandran, Sudhanshu Vrati, Arup Banerjee

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Background: Long non-coding RNAs (lncRNAs) are the important regulators of gene expression and play important role in viral replication and disease progression. The role of lncRNA genes in the pathogenesis of Dengue virus-mediated pathogenesis is currently unknown. Methods: To gain additional insights, we utilized an unbiased RNA sequencing followed by in silico analysis approach to identify the differentially expressed lncRNA and genes that are associated with dengue disease progression. Further, we focused our study on lncRNAs NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) as it was found to be differentially expressed in PBMC of dengue infected patients. Results: The expression of lncRNAs NEAT1, as compared to dengue infection (DI), was significantly down-regulated as the patients developed the complication. Moreover, pairwise analysis on follow up patients confirmed that suppression of NEAT1 expression was associated with rapid fall in platelet count in dengue infected patients. Severe dengue patients (DS) (n=18; platelet count < 20K) when recovered from infection showing high NEAT1 expression as it observed in healthy donors. By co-expression network analysis and subsequent validation, we revealed that coding gene; IFI27 expression was significantly up-regulated in severe dengue cases and negatively correlated with NEAT1 expression. To discriminate DI from dengue severe, receiver operating characteristic (ROC) curve was calculated. It revealed sensitivity and specificity of 100% (95%CI: 85.69 – 97.22) and area under the curve (AUC) = 0.97 for NEAT1. Conclusions: Altogether, our first observations demonstrate that monitoring NEAT1and IFI27 expression in dengue patients could be useful in understanding dengue virus-induced disease progression and may be involved in pathophysiological processes.

Keywords: dengue, lncRNA, NEAT1, transcriptome

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Authors: Yazun Jarrar

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Smoking has effect on platelet aggregation and the activity of anti-platelet drugs. The chemical 20-hydroxyeicosatetraenoic acid (20-HETE) is a cardiotoxic arachidonic acid metabolite which increases platelet aggregation. In this study, we investigated the influence of cigarette smoking on 20-HETE levels and protein expression of 20-HETE producing enzyme CYP4A11 in isolated platelets from smoker and non-smoker volunteers. The protein expression and 20-HETE levels were analyzed using immunoblot and High-Performance Liquid Chromatography with Mass Spectrometry (HPL-MS) assays. The results showed that 20-HETE level was higher significantly among smokers than non-smokers (t-test, p-value<0.05). The protein expression of CYP4A11 was significantly higher (t-test, p-value<0.05) among the platelets of smokers. We concluded that cigarette smoking increased the level of platelet activator 20-HETE through increasing the protein expression of CYP4A11. These findings may increase the understanding of smoking-drug interaction during antiplatelets therapy.

Keywords: smoking, 20-HETE, CYP4A11, platelet

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Authors: Chaiwat Boonkaewwan, Anutian Suklek, Jatuporn Rattanasrisomporn, Autchara Kayan

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Toll-like receptors (TLR) are conserved microbial sensing receptors located on cell surface that are able to detect different pathogens. The aim of the present study is to examine the expression of TLR gene in peripheral blood mononuclear cell of Betong (KU line) chicken. Blood samples were collected from healthy 12 Betong (KU line) chicken. PBMCs were isolated and maintained in RPMI1640 with 10% FBS, penicillin and streptomycin. Cell viability was determined by trypan blue dye exclusion test. The expression of TLRs gene was investigated by polymerase chain reaction (PCR) technique. Results showed that PBMCs viability from Betong (KU line) chicken was 95.38 ± 1.06%. From the study of TLRs gene expression, results indicated that there are expressions of TLR1.1 TLR1.2 TLR2.1 TLR2.2 TLR3 TLR4 TLR5 TLR 7 TLR15 and TLR21 in PBMCs of Betong (KU line) chicken. In conclusion, PBMCs isolated from blood of Betong (KU line) chicken had a high cell viability ( > 95%). The expression of TLRs in chicken was all found in PBMCs, which indicated that PBMC isolated from the blood of Betong (KU line) chicken can be used as an in vitro immune responses study.

Keywords: toll-like receptor, Betong (KU line) chicken, peripheral blood mononuclear cells

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Authors: Y. C. Khoo, W. T. Lai

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The single orifice hydraulic flat spray nozzle was evaluated with two global imaging techniques to characterize various aspects of the resulting spray. The two techniques were high resolution flow visualization and Particle Image Velocimetry (PIV). A CCD camera with 29 million pixels was used to capture shadowgraph images to realize ligament formation and collapse as well as droplet interaction. Quantitative analysis was performed to give the sizing information of the droplets and ligaments. This camera was then applied with a PIV system to evaluate the overall velocity field of the spray, from nozzle exit to droplet discharge. PIV images were further post-processed to determine the inclusion angle of the spray. The results from those investigations provided significant quantitative understanding of the spray structure. Based on the quantitative results, detailed understanding of the spray behavior was achieved.

Keywords: spray, flow visualization, PIV, shadowgraph, quantitative sizing, velocity field

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Authors: Muhammad Haroon, LI Xiangju

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C. communis L. is an important weed of many crop, but very little information about the adaptation strategies of C. communis L. biotypes under drought stress. We investigated five biotypes of C. communis L under drought stress to identify the adaptation mechanism. The expression of drought stress related genes (DRS1, EREB and HRB1) was up-regulated in biotypes, while in some biotypes their expression was down regulated. All five biotypes can thus regulate water balance to consume less water to maintain their status under drought stress condition. This result concluded that C. communis L. biotypes can survive longer under drought stress condition. Weed scientist should seek more effective management strategies to deal with C. communis L.

Keywords: C. communis, biotypes, drought stress, gene expression

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Authors: Changfeng Fu

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Sustainable urban development has been widely accepted a common sense in the modern urban planning and design. However, the measurement and assessment of urban sustainability, especially the quantitative assessment have been always an issue obsessing planning and design professionals. This paper will present an on-going research on the principles and technologies to develop a quantitative urban sustainability assessment principles and techniques which aim to integrate indicators, geospatial and geo-reference data, and assessment techniques together into a mechanism. It is based on the principles and techniques of geospatial analysis with GIS and statistical analysis methods. The decision-making technologies and methods such as AHP and SMART are also adopted to address overall assessment conclusions. The possible interfaces and presentation of data and quantitative assessment results are also described. This research is based on the knowledge, situations and data sources of UK, but it is potentially adaptable to other countries or regions. The implementation potentials of the mechanism are also discussed.

Keywords: urban sustainability assessment, quantitative analysis, sustainability indicator, geospatial data, big data

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Authors: Massimiliano Biagini, Marco Spinsanti, Gabriella De Angelis, Sara Tomei, Ilaria Ferlenghi, Maria Scarselli, Alessia Biolchi, Alessandro Muzzi, Brunella Brunelli, Silvana Savino, Marzia M. Giuliani, Isabel Delany, Paolo Costantino, Rino Rappuoli, Vega Masignani, Nathalie Norais

Abstract:

The gram-negative bacterium Neisseria meningitidis serogroup B (MenB) is an exclusively human pathogen representing the major cause of meningitides and severe sepsis in infants and children but also in young adults. This pathogen is usually present in the 30% of healthy population that act as a reservoir, spreading it through saliva and respiratory fluids during coughing, sneezing, kissing. Among surface-exposed protein components of this diplococcus, factor H binding protein is a lipoprotein proved to be a protective antigen used as a component of the recently licensed Bexsero vaccine. fHbp is a highly variable meningococcal protein: to reflect its remarkable sequence variability, it has been classified in three variants (or two subfamilies), and with poor cross-protection among the different variants. Furthermore, the level of fHbp expression varies significantly among strains, and this has also been considered an important factor for predicting MenB strain susceptibility to anti-fHbp antisera. Different methods have been used to assess fHbp expression on meningococcal strains, however, all these methods use anti-fHbp antibodies, and for this reason, the results are affected by the different affinity that antibodies can have to different antigenic variants. To overcome the limitations of an antibody-based quantification, we developed a quantitative Mass Spectrometry (MS) approach. Selected Reaction Monitoring (SRM) recently emerged as a powerful MS tool for detecting and quantifying proteins in complex mixtures. SRM is based on the targeted detection of ProteoTypicPeptides (PTPs), which are unique signatures of a protein that can be easily detected and quantified by MS. This approach, proven to be highly sensitive, quantitatively accurate and highly reproducible, was used to quantify the absolute amount of fHbp antigen in total extracts derived from 105 clinical isolates, evenly distributed among the three main variant groups and selected to be representative of the fHbp circulating subvariants around the world. We extended the study at the genetic level investigating the correlation between the differential level of expression and polymorphisms present within the genes and their promoter sequences. The implications of fHbp expression on the susceptibility of the strain to killing by anti-fHbp antisera are also presented. To date this is the first comprehensive fHbp expression profiling in a large panel of Neisseria meningitidis clinical isolates driven by an antibody-independent MS-based methodology, opening the door to new applications in vaccine coverage prediction and reinforcing the molecular understanding of released vaccines.

Keywords: quantitative mass spectrometry, Neisseria meningitidis, vaccines, bexsero, molecular epidemiology

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Authors: Mhaned Oubounyt, Jan Baumbach

Abstract:

Differences in co-expression networks between two or multiple cells (sub)types across conditions is a pressing problem in single-cell RNA sequencing (scRNA-seq). A key challenge is to define those co-variations that differ between or among cell types and/or conditions and phenotypes to examine small regulatory networks that can explain mechanistic differences. To this end, we developed SCANet, an all-in-one Python package that uses state-of-the-art algorithms to facilitate the workflow of a combined single-cell GCN (Gene Correlation Network) and GRN (Gene Regulatory Networks) pipeline, including inference of gene co-expression modules from scRNA-seq, followed by trait and cell type associations, hub gene detection, co-regulatory networks, and drug-gene interactions. In an example case, we illustrate how SCANet can be applied to identify regulatory drivers behind a cytokine storm associated with mortality in patients with acute respiratory illness. SCANet is available as a free, open-source, and user-friendly Python package that can be easily integrated into systems biology pipelines.

Keywords: single-cell, co-expression networks, drug-gene interactions, co-regulatory networks

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Authors: Kun Chun, Jin-Chun Heo, Sang-Han Lee

Abstract:

Asthma is a chronic airway inflammatory disease characterized by the infiltration of inflammatory cells and tissue remodeling. In this study, we examined the anti-asthmatic activity of a derivative of L-allo threonine by in vitro and in vivo anti-asthmatic assays. Ovalbumin (OVA)-induced C57BL/6 mice were used to analyze lung inflammation and cytokine expressions for exhibiting anti-atopic activity of the derivative. LX519290, a derivative of L-allo threonine, induced an increased IFN-γ and a decreased IL-10 mRNA level. This compound exhibited potent anti-asthmatic activity by decreasing immune cell infiltration in the lung, and IL-4 and IL-13 cytokine levels in the serum of OVA-induced mice. These results indicated that chronic airway injury was decreased by LX519290. We also assessed that LX519290 inhibits infiltration of immune cell, mucus release and cytokine expression in an in vivo model. Our results collectively suggest that the L-allo threonine is effective in alleviating asthmatic symptoms by treating inflammatory factors in the lung.

Keywords: asthma, L -allo threonine, LX519290, mice

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Authors: Akterono Budiyati, Gita Kusumo, Teguh Putra, Fritzie Rexana, Antonius Kurniawan, Aru Sudoyo, Ahmad Utomo, Andi Utama

Abstract:

The presence of the programmed death ligand-1 (PD-L1) has been used in multiple clinical trials and approved as biomarker for selecting patients more likely to respond to immune checkpoint inhibitors. However, the expression of PD-L1 is regulated in different ways, which leads to a different significance of its presence. Positive PD-L1 within tumors may result from two mechanisms, induced PD-L1 expression by T-cell presence or genetic mechanism that lead to constitutive PD-L1 expression. Amplification of PD-L1 genes was found as one of genetic mechanism which causes an increase in PD-L1 expression. In case of colorectal cancer (CRC), targeting immune checkpoint inhibitor has been recommended for patients with microsatellite instable (MSI). Although the correlation between PD-L1 expression and MSI status has been widely studied, so far the precise mechanism of PD-L1 gene activation in CRC patients, particularly in MSI population have yet to be clarified. In this present study we have profiled 61 archived formalin fixed paraffin embedded CRC specimens of patients from Medistra Hospital, Jakarta admitted in 2010 - 2016. Immunohistochemistry was performed to measure expression of PD-L1 in tumor cells as well as MSI status using antibodies against PD-L1 and MMR (MLH1, MSH2, PMS2 and MSH6), respectively. PD-L1 expression was measured on tumor cells with cut off of 1% whereas loss of nuclear MMR protein expressions in tumor cells but not in normal or stromal cells indicated presence of MSI. Subset of PD-L1 positive patients was then assessed for copy number variations (CNVs) using single Tube TaqMan Copy Number Assays Gene CD247PD-L1. We also observed KRAS mutation to profile possible genetic mechanism leading to the presence or absence of PD-L1 expression. Analysis of 61 CRC patients revealed 15 patients (24%) expressed PD-L1 on their tumor cell membranes. The prevalence of surface membrane PD-L1 was significantly higher in patients with MSI (87%; 7/8) compared to patients with microsatellite stable (MSS) (15%; 8/53) (P=0.001). Although amplification of PD-L1 gene was not found among PD-L1 positive patients, low-level amplification of PD-L1 gene was commonly observed in MSS patients (75%; 6/8) than in MSI patients (43%; 3/7). Additionally, we found 26% of CRC patients harbored KRAS mutations (16/61), so far the distribution of KRAS status did not correlate with PD-L1 expression. Our data suggest genetic mechanism through amplification of PD-L1 seems not to be the mechanism underlying upregulation of PD-L1 expression in CRC patients. However, further studies are warranted to confirm the results.

Keywords: colorectal cancer, gene amplification, microsatellite instable, programmed death ligand-1

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Authors: Saad Al-Shibli, Nasser Amjad, Muna Al Kubaisi, Norra Harun, Shaikh Mizan

Abstract:

Leptin is a multifunctional hormone produced mainly by adipocyte. Leptin and its receptor have long been found associated with breast cancer. The main aim of this study is to investigate the correlation between Leptin/Leptin receptor and the clinicopathological features of breast cancer. Blood samples for ELISA, tissue samples from tumors and adjacent breast tissue were taken from 51 women with breast cancer with a control group of 40 women with a negative mammogram. Leptin and Leptin receptor in the tissues were estimated by immunohistochemistry (IHC). They were localized at the subcellular level by immunocytochemistry using transmission electron microscopy (TEM). Our results showed significant difference in serum leptin level between control and the patient group, but no difference between pre and post-operative serum leptin levels in the patient group. By IHC, we found that the majority of the breast cancer cells studied, stained positively for leptin and leptin receptors with co-expression of leptin and its receptors. No significant correlation was found between leptin/leptin receptors expression with the race, menopausal status, lymph node metastasis, estrogen receptor expression, progesterone receptor expression, HER2 expression and tumor size. Majority of the patients with distant metastasis were associated with high leptin and leptin receptor expression. TEM views both Leptin and Leptin receptor were found highly concentrated within and around the nucleus of the cancer breast cells, indicating nucleus is their principal seat of actions while the adjacent breast epithelial cells showed that leptin gold particles are scattered all over the cell with much less than that of the cancerous cells. However, presence of high concentration of leptin does not necessarily prove its over-expression, because it could be internalized from outside by leptin receptor in the cells. In contrast, leptin receptor is definitely over-expressed in the ductal breast cancer cells. We conclude that reducing leptin levels, blocking its downstream tissue specific signal transduction, and/or blocking the upstream leptin receptor pathway might help in prevention and therapy of breast cancer.

Keywords: breast cancer, expression, leptin, leptin receptors

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Authors: Anshu Bahl, Saroj Kaler, Shivani Gupta, S B Ray

Abstract:

Background: Somatostatin is an endogenous regulatory neuropeptide. Somatostatin and its analogues play an important role in neuropathic and inflammatory pain. Neuropeptide Y is extensively distributed in the mammalian nervous system. NPY has an important role in blood pressure, circadian rhythm, obesity, appetite and memory. The purpose was to investigate somatostatin and NPY expression in dorsal root ganglia during pain. The plantar incision model in rats is similar to postoperative pain in humans. Methods: 24 adult male Sprague dawley rats were distributed randomly into two groups – Control (n=6) and incision (n=18) groups. Using Hargreaves apparatus, thermal hyperalgesia behavioural test for nociception was done under basal condition and after surgical incision in right hind paw at different time periods (day 1, 3 and 5). The plantar incision was performed as per standard protocol. Perfusion was done using 4% paraformaldehyde followed by extraction of dorsal root ganglia at L4 level. The tissue was processed for immunohistochemical localisation for somatostatin and neuropeptide Y. Results: Post incisional groups (day 1, 3 and 5) exhibited significant decrease of paw withdrawal latency as compared to control groups. Somatostatin expression was noted under basal conditions. It decreased on day 1, but again gradually increased on day 3 and further on day five post incision. The expression of Neuropeptide Y was noted in the cytoplasm of dorsal root ganglia under basal conditions. Compared to control group, expression of neuropeptide Y decreased on day one after incision, but again gradually increased on day 3. Maximum expression was noted on day five post incision. Conclusion: Decrease in paw withdrawal latency indicated nociception, particularly on day 1. In comparison to control, somatostatin and NPY expression was decreased on day one post incision. This could be correlated with increased axoplasmic flow towards the spinal cord. Somatostatin and NPY expression was maximum on day five post incision. This could be due to decreased migration from the site of synthesis towards the spinal cord.

Keywords: dorsal root ganglia, neuropeptide y, postoperative pain, somatostatin

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Authors: Yahia I. Mohamed, Ahmed I. Marzouk, Mohamed A. Yacout

Abstract:

Aim of this work was to study the genetic basis for oil accumulation in olive fruit via tracking DGAT2 (Diacylglycerol acyltransferase type-2) gene in three Egyptian Origen Olive cultivars namely Toffahi, Hamed and Maraki using molecular marker techniques and bioinformatics tools. Results illustrate that, firstly: specific genomic band of Maraki cultivars was identified as DGAT2 (Diacylglycerol acyltransferase type-2) and identical for this gene in Olea europaea with 100 % of similarity. Secondly, differential genomic band of Maraki cultivars which produced from RAPD fingerprinting technique reflected predicted distinguished sequence which identified as DGAT2 (Diacylglycerol acyltransferase type-2) in Fragaria vesca subsp. Vesca with 76% of sequential similarity. Third and finally, specific genomic specific band of Hamed cultivars was indentified as two fragments, 1-Olea europaea cultivar Koroneiki diacylglycerol acyltransferase type 2 mRNA, complete cds with two matches regions with 99% or 2-PREDICTED: Fragaria vesca subsp. vesca diacylglycerol O-acyltransferase 2-like (LOC101313050), mRNA with 86% of similarity.

Keywords: Olea europaea, fingerprinting, diacylglycerol acyltransferase type-2 (DGAT2), Egypt

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Authors: Seong Gu Hwang, Young Kyoon Oh, Joseph F. dela Cruz

Abstract:

Marbling, or intramuscular fat, has been consistently identified as one of the top beef quality problems. Intramuscular adipocytes distribute throughout the perimysial connective tissue of skeletal muscle and are the major site for the deposition of intramuscular fat, which is essential for the eating quality of meat. The stromal vascular fraction of the skeletal muscle contains progenitor cells that can be enhanced to differentiate to adipocytes and increase intramuscular fat. Primary cultures of bovine intramuscular stromal vascular cells were used in this study to elucidate the effects of extracellular calcium and retinoic acid concentration on adipocyte differentiation. Cell viability assay revealed that even at different concentrations of calcium and retinoic acid, there was no significant difference on cell viability. Monitoring of the adipocyte differentiation showed that bovine intramuscular stromal vascular cells cultured in a low concentration of extracellular calcium and retinoic acid had a better degree of fat accumulation. The mRNA and protein expressions of PPARγ, C/EBPα, SREBP-1c and aP2 were analyzed and showed a significant upregulation upon the reduction in the level of extracellular calcium and retinoic acid. The upregulation of these adipogenic related genes means that the decreasing concentration of calcium and retinoic acid is able to stimulate the adipogenic differentiation of bovine intramuscular stromal vascular cells. To further elucidate the effect of calcium, the expression level of calreticulin was measured. Calreticulin which is known to be an inhibitor of PPARγ was down regulated by the decreased level of calcium and retinoic acid in the culture media. The same tendency was observed on retinoic acid receptors RARα and CRABP-II. These receptors are recognized as adipogenic inhibitors, and the downregulation of their expression allowed a better level of differentiation in bovine intramuscular stromal vascular cells. In conclusion, data show that decreasing the level of extracellular calcium and retinoic acid can significantly promote adipogenesis in intramuscular stromal vascular cells of Hanwoo beef cattle. These findings may provide new insights in enhancing intramuscular adipogenesis and marbling in beef cattle.

Keywords: calcium, calreticulin, hanwoo beef, retinoic acid

Procedia PDF Downloads 279