Search results for: gene expression profiles

Authors: Mudasir Ahmad Syed, Raashid Ahmd Wani, Mashooq Ahmad Dar, Uneeb Urwat, Riaz Ahmad Shah, Nazir Ahmad Ganai

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Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a primary avian pathogen responsible for severe intestinal pathology in younger chickens and economic losses. However, the Salmonella Typhimurium is also able to cause infection in humans, described by typhoid fever and acute gastro-intestinal disease. A study was conducted at days to investigate pathological, histopathological, haemato-biochemical, immunological and expression kinetics of NRAMP (natural resistance associated macrophage protein) gene family (NRAMP1 and NRAMP2) in broiler chickens following experimental infection of Salmonella Typhimurium at 0,1,3,5,7,9,11,13 and 15 days respectively. Infection was developed in birds through oral route at 2×108 CFU/ml. Clinical symptoms appeared 4 days post infection (dpi) and after one-week birds showed progressive weakness, anorexia, diarrhea and lowering of head. On postmortem examination, liver showed congestion, hemorrhage and necrotic foci on surface, while as spleen, lungs and intestines revealed congestion and hemorrhages. Histopathological alterations were principally observed in liver in second week post infection. Changes in liver comprised of congestion, areas of necrosis, reticular endothelial hyperplasia in association with mononuclear cell and heterophilic infiltration. Hematological studies confirm a significant decrease (P<0.05) in RBC count, Hb concentration and PCV. White blood cell count showed significant increase throughout the experimental study. An increase in heterophils was found up to 7dpi and a decreased pattern was observed afterwards. Initial lymphopenia followed by lymphocytosis was found in infected chicks. Biochemical studies showed a significant increase in glucose, AST and ALT concentration and a significant decrease (P<0.05) in total protein and albumin level in the infected group. Immunological studies showed higher titers of IgG in infected group as compared to control group. The real time gene expression of NRAMPI and NRAMP2 genes increased significantly (P<0.05) in infected group as compared to controls. The peak expression of NRAMP1 gene was seen in liver, spleen and caecum of infected birds at 3dpi, 5dpi and 7dpi respectively, while as peak expression of NRAMP2 gene in liver, spleen and caecum of infected chicken was seen at 9dpi, 5dpi and 9dpi respectively. This study has role in diagnostics and prognostics in the poultry industry for the detection of salmonella infections at early stages of poultry development.

Keywords: biochemistry, histopathology, NRAMP, poultry, real time expression, Salmonella Typhimurium

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Authors: Mahbuba Rahman, Sabri Boughorbel, Scott Presnell, Charlie Quinn, Darawan Rinchai, Damien Chaussabel, Nico Marr

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Collections of large-scale datasets made available in public repositories can be used to identify and fill gaps in biomedical knowledge. But first, these data need to be made readily accessible to researchers for analysis and interpretation. Here a collection of transcriptome datasets was made available to investigate the functional programming of human hematopoietic cells in early life. Thirty two datasets were retrieved from the NCBI Gene Expression Omnibus (GEO) and loaded in a custom, interactive web application called the Gene Expression browser (GXB), designed for visualization and query of integrated large-scale data. Multiple sample groupings and gene rank lists were created based on the study design and variables in each dataset. Web links to customized graphical views can be generated by users and subsequently be used to graphically present data in manuscripts for publication. The GXB tool also enables browsing of a single gene across datasets, which can provide information on the role of a given molecule across biological systems. The dataset collection is available online. As a proof-of-principle, one of the datasets (GSE25087) was re-analyzed to identify genes that are differentially expressed by regulatory T cells in early life. Re-analysis of this dataset and a cross-study comparison using multiple other datasets in the above mentioned collection revealed that PMCH, a gene encoding a precursor of melanin-concentrating hormone (MCH), a cyclic neuropeptide, is highly expressed in a variety of other hematopoietic cell types, including neonatal erythroid cells as well as plasmacytoid dendritic cells upon viral infection. Our findings suggest an as yet unrecognized role of MCH in immune regulation, thereby highlighting the unique potential of the curated dataset collection and systems biology approach to generate new hypotheses which can be tested in future mechanistic studies.

Keywords: early-life, GEO datasets, PMCH, interactive query, systems biology

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Authors: Tania Tzong, Chao-Yi Teng, Tzong-Yuan Wu

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Currently, Chikungunya virus (CHIKV) transmitted to humans by Aedes mosquitoes has distributed from Africa to Southeast Asia, South America, and South Europe. However, little is known about the antigenic targets for immunity, and there are no licensed vaccines or specific antiviral treatments for the disease caused by CHIKV. Baculovirus has been recognized as a novel vaccine vector with attractive characteristic features of an optional vaccine delivery vehicle. This approach provides the safety and efficacy of CHIKV vaccine. In this study, bi-cistronic recombinant baculoviruses vAc-CMV-CHIKV26S-Rhir-EGFP and vAc-CMV-pH-CHIKV26S-Lir-EGFP were produced. Both recombinant baculovirus can express EGFP reporter gene in insect cells to facilitate the recombinant virus isolation and purification. Examination of vAc-CMV-CHIKV26S-Rhir-EGFP and vAc-CMV-pH-CHIKV26S-Lir-EGFP showed that this recombinant baculovirus could induce syncytium formation in insect cells. Unexpectedly, the immunofluorescence assay revealed the expression of E1 and E2 of CHIKV structural proteins in insect cells infected by vAc-CMV-CHIKV26S-Rhir-EGFP. This result may imply that the CMV promoter can induce the transcription of CHIKV26S in insect cells. There are also E1 and E2 expression in mammalian cells transduced by vAc-CMV-CHIKV26S-Rhir-EGFP and vAc-CMV-pH-CHIKV26S-Lir-EGFP. The expression of E1 and E2 proteins of insect and mammalian cells was validated again by Western blot analysis. The vector construction with dual tandem promoters, which is polyhedrin and CMV promoter, has higher expression of the E1 and E2 of CHIKV structural proteins than the vector construction with CMV promoter only. Most of the E1 and E2 proteins expressed in mammalian cells were glycosylated. In the future, the expression of structural proteins of CHIKV in mammalian cells is expected can form virus-like particle, so it could be used as a vaccine for chikungunya virus.

Keywords: chikungunya virus, virus-like particle, vaccines, baculovirus expression vector system

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Authors: Arfan Ali, Idrees Ahmad Nasir

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The critical evaluation of tolerance in tomato plants against the induced saline soil were assessed by transcript analysis of genes coding for products potentially involved in stress tolerance. A reverse transcriptase PCR experiment was performed with Hsp90-1, MT2, and GR1like protein genes using RNA isolated from different tissues of tomato plants. Four strains of Bacillus magisterium were inoculated with 100 Mm & 200 Mm concentrations of salt. Eleven treatments each ten replica pots were installed in green house experiment and the parameters taken into account were morphological (length, weight, number of leaves, leaf surface area), chemical (anthocyanin, chlorophyll-a, chlorophyll-b, carotenoids) and biological (gene expression). Results bare a response i.e. highest response of MT2 like gene was at 24 hpi and the highest levels of GR1 like protein transcript accumulation were detected at 36 hpi. The chemical and morphological parameters at diverse salt concentrations bequeath superlative response amongst strains which candidly flank on Zm7 and Zm4. Therefore, Bacillus magisterium Zm7 strains and somehow Zm4 strain can be used in saline condition to make plants tolerant. The overall performance of strains Zm7, Zm6, and Zm4 was found better for all studied traits under salt stress conditions. Significant correlations among traits root length, shoot length, number of leaves, leaf surface area, carotenoids, anthocyanin, chlorophyll-a and chlorophyll-b were found and suggested that the salt tolerance in tomato may be improved through the use of PGPR strains.

Keywords: Bacillus magisterium, gene expression glutathione reductase, metallothionein, PGPR, Rhizobacteria, saline

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Authors: Rachel Matar, Maxime Merheb

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Chemical drugs have been used for many centuries as the only way to cure diseases until the novel gene therapy has been created in 1960. Gene therapy is based on the insertion, correction, or inactivation of genes to treat people with genetic illness (1). Gene therapy has made wonders in Parkison’s, Alzheimer and multiple sclerosis. In addition to great promises in the healing of deadly diseases like many types of cancer and autoimmune diseases (2). This method implies the use of recombinant DNA technology with the help of different viral and non-viral vectors (3). It is nowadays used in somatic cells as well as embryos and gametes. Beside all the benefits of gene therapy, this technique is deemed by some opponents as an ethically unacceptable treatment as it implies playing with the genes of living organisms.

Keywords: gene therapy, genetic disease, cancer, multiple sclerosis

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Authors: Daina Jonkus, Liga Paura, Tatjana Sjakste, Kristina Dokane

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The aim of this study was to analyse PRKAG3 and RYR1 gene and genotypes frequencies in Latvian White pigs’ breed. Genotypes of RYR1 gene two loci (rs196953058 and rs323041392) in 89 exon and PRKAG3 gene two loci (rs196958025 and rs344045190) in gene promoter were detected in 103 individuals of Latvian white pigs’ breed. Analysis of RYR1 gene loci rs196953058 shows all individuals are homozygous by T allele and all animals are with genotypes TT, its mean - in 2769 position is Phenylalanine. Analysis of RYR1 gene loci rs323041392 shows all individuals are homozygous by G allele and all animals are with genotypes GG, its mean - in 4119 positions is Asparagine. In loci rs196953058 and rs323041392, there were no gene polymorphisms. All analysed individuals by two loci rs196953058-rs323041392 have TT-GG genotypes or Phe-Asp amino acids. In PRKAG3 gene loci rs196958025 and rs344045190 there was gene polymorphisms. In both loci frequencies for A allele was higher: 84.6% for rs196958025 and 73.0% for rs344045190. Analysis of PRKAG3 gene loci rs196958025 shows 74% of individuals are homozygous by An allele and animals are with genotypes AA. Only 4% of individuals are homozygous by G allele and animals are with genotypes GG, which is associated with pale meat colour and higher drip loss. Analysis of PRKAG3 gene loci rs344045190 shows 46% of individuals are homozygous with genotypes AA and 54% of individuals are heterozygous with genotypes AG. There are no individuals with GG genotypes. According to the results, in Latvian white pigs population there are no rs344435545 (RYR1 gene) CT heterozygous or TT recessive homozygous genotypes, which is related to the meat quality and pigs’ stress syndrome; and there are 4% rs196958025 (PRKAG3 gene) GG recessive homozygote genotypes, which is related to the meat quality. Acknowledgment: the investigation is supported by VPP 2014-2017 AgroBioRes Project No. 3 LIVESTOCK.

Keywords: genotype frequencies, pig, PRKAG3, RYR1

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Authors: Hamid Mustafa, Adeela Ajmal, Kim EuiSoo, Noor-ul-Ain

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World wild, buffalo production is considered as most important component of food industry. Efficient buffalo production is related with reproductive performance of this species. Lack of knowledge of reproductive efficiency and its related genes in buffalo species is a major constraint for sustainable buffalo production. In this study, we performed some bioinformatics analysis on Follicle Stimulating Hormone Receptor (FSHR) gene and explored the possible relationship of this gene among different buffalo breeds and with other farm animals. We also found the evolution pattern for this gene among these species. We investigate CDS lengths, Stop codon variation, homology search, signal peptide, isoelectic point, tertiary structure, motifs and phylogenetic tree. The results of this study indicate 4 different motif in this gene, which are Activin-recp, GS motif, STYKc Protein kinase and transmembrane. The results also indicate that this gene has very close relationship with cattle, bison, sheep and goat. Multiple alignment (MA) showed high conservation of motif which indicates constancy of this gene during evolution. The results of this study can be used and applied for better understanding of this gene for better characterization of Follicle Stimulating Hormone Receptor (FSHR) gene structure in different farm animals, which would be helpful for efficient breeding plans for animal’s production.

Keywords: buffalo, FSHR gene, bioinformatics, production

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Authors: Komal Vig, Syamala Soumyakrishnan, Yadav Baral

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Bypass surgery, using the autologous vein has been one of the most effective treatments for cardiovascular diseases (CVD). More recently tissue engineering including engineered vascular grafts to synthesize blood vessels is gaining usage. Dacron and ePTFE has been employed for vascular grafts, however, these does not work well for small diameter grafts (<6 mm) due to intimal hyperplasia and thrombosis. In the present study PTFE was treated with LTP to improve the endothelialization of intimal surface of graft. Scaffolds were also modified with polyvinylpyrrolidone coated silver nanoparticles (Ag-PVP) and the antimicrobial peptides, p753 and p359. Human umbilical vein endothelial cells (HUVEC) were plated on the developed scaffolds and cell proliferation was determined by the MTT assay. Cells attachment on scaffolds was visualized by microscopy. mRNA expressions levels of different cell markers were investigated using quantitative real-time PCR (qPCR). X ray photoelectron spectroscopic confirmed the introduction of oxygenated functionalities from LTP air plasma. Microscopic and MTT assays indicated increase in cell viability in LTP treated scaffolds. Gene expression studies shows enhanced expression of cell adhesion marker Integrin- α 5 gene after LTP treatment. The KB test displayed a zone of inhibition for Ag-PVP, p753 and p359 of 19mm, 14mm, and 12mm respectively. To determine toxicity of antimicrobial agents to cells, MTT Assay was performed using HEK293 cells. MTT Assay exhibited that Ag-PVP and the peptides were non-toxic to cells at 100μg/mL and 50μg/mL, respectively. Live/dead analysis and plate count of treated bacteria exhibited bacterial inhibition on develop scaffold compared to non-treated scaffold. SEM was performed to analyze the structural changes of bacteria after treatment with antimicrobial agents. Gene expression studies were conducted on RNA from bacteria treated with Ag-PVP and peptides using qRT-PCR. Based on our initial results, more scaffolds alternatives will be developed and investigated for cell growth and vascularization studies.

Keywords: low temperature plasma, vascular graft, HUVEC cells, antimicrobial

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Authors: Sevil Yildiz

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Audio visual communication is a type of collective expression. Collective expression activity informs the masses, gives direction to opinions and establishes public opinion. Due to these characteristics, audio visual communication must be subjected to special restrictions. This has been stipulated in both the Constitution and the European Human Rights Agreement. This paper aims to review freedom of expression and its restriction in audio visual media. For this purpose, the authorisation of the Radio and Television Supreme Council to impose sanctions as an independent administrative authority empowered to regulate the field of audio visual communication has been reviewed with regard to freedom of expression and its limits.

Keywords: audio visual media, freedom of expression, its limits, radio and television supreme council

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Authors: Shahram Nanekarania, Majid Goodarzia

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Calpastatin and callipyge have been known as one of the candidate genes in meat quality and quantity. Calpastatin gene has been located to chromosome 5 of sheep and callipyge gene has been localized in the telomeric region on ovine chromosome 18. The objective of this study was identification of calpastatin and callipyge genes polymorphism and analysis of genotype structure in population of Lori sheep kept in Iran. Blood samples were taken from 120 Lori sheep breed and genomic DNA was extracted by salting out method. Polymorphism was identified using the PCR-RFLP technique. The PCR products were digested with MspI and FaqI restriction enzymes for calpastatin gene and callipyge gene, respectively. In this population, three patterns were observed and AA, AB, BB genotype have been identified with the 0.32, 0.63, 0.05 frequencies for calpastatin gene. The results obtained for the callipyge gene revealed that only the wild-type allele A was observed, indicating that only genotype AA was present in the population under consideration.

Keywords: polymorphism, calpastatin, callipyge, PCR-RFLP, Lori sheep

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Authors: Hye Kyung Kim, Myung-Gyou Kim, Kang-Hyun Leem

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Postmenopausal osteoporosis is characterized by low bone density which leads to increased bone fragility and greater susceptibility to fracture. Current treatments for osteoporosis are dominated by drugs that inhibit bone resorption although they also suppress bone formation that may contribute to pathogenesis of osteonecrosis. To restore the extensive bone loss, there is a great need for anabolic treatments that induce osteoblasts to build new bone. Pre-osteoblastic cells produce proteins of the extra-cellular matrix, including type I collagen at first, and then to successively produce alkaline phosphatase (ALP) and osteocalcin during differentiation to osteoblasts. Finally, osteoblasts deposit calcium. Present study investigated the effects of egg yolk peptide (EYP) on osteogenic activities and bone matrix gene expressions in human osteoblastic MG-63 cells. The effects of EYP on cell proliferation, alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization were measured. The expression of osteogenic genes including COL1A1 (collagen, type I, alpha 1), ALP, BGLAP (osteocalcin), and SPP1 (secreted phosphoprotein 1, osteopontin) were measured by quantitative realtime PCR. EYP dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. Furthermore, COL1A1, ALP, and SPP1 gene expressions were increased by EYP treatment. Present study suggested that EYP treatment enhanced osteogenic activities and increased bone matrix osteogenicgenes. These results could provide a mechanistic explanation for the bone-strengthening effects of EYP.

Keywords: egg yolk peptide, osteoblastic MG-63 cells, alkaline phosphatase, collagen synthesis, osteogenic genes, COL1A1, osteocalcin, osteopontin

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Authors: Ricardo de Oliveira Orsi, Aline Astolfi, Daniel Diego Mendes, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, Yan Souza Lima, Juliana Lunardi, Giovanna do Padro Ribeiro, Samir Moura Kadri

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NECTAR Brazilian group (Center of Education, Science, and Technology in Rational Beekeeping) conducted studies on the pesticides honey bees effects using the transcriptome sequencing (RNA-Seq) analyzes for gene expression studies. In this way, we analyzed the effects of Pyraclostrobin and Fipronil on the honey bees with 21 old-days (forager) in laboratory conditions. For this, frames containing sealed brood were removed from the beehives and maintenance on the stove (32°C and 75% humidity) until the bees were born. So, newly emerged workers were marked on the pronotum with a non-toxic pen and reintroduced into their original hives. After 21 days, 120 marked bees were collected with an entomological forces and immediately stored in Petri dishes, perforated to ensure ventilation, and kept fasted for 3 hours. These honeybees were exposed to food contaminated or not with the sublethal dose of Pyraclostrobin (850 ppb/bee) or Fipronil (2.5 ppb/bee). After four hours of exposure, 15 bees from each treatment were referred to transcriptome analysis. Total RNA analysis was extracted from the brain pools (03 brains per pool) using the TRIzol® reagent protocol according to the manufacturer's instructions. cDNA libraries were constructed, and the FASTQC program was used to check adapter content and assess the quality of raw reads. Differential expression analysis was performed with the DESeq2 package. Genes that had an adjusted value of less than 0.05 were considered to be significantly up-regulated. Regarding the Pyraclostrobin, alterations were observed in the pattern of 17 gene related to of antioxidant system, cellular respiration, glucose metabolism, and regulation of juvenile hormone and the hormone insulin. Glyphosate altered the 10 gene related to the digestive system, exoskeleton composition, vitamin E transport, and antioxidant system. The results indicate that the necessity of studies using the sublethal doses to evaluate the pesticides uses and risks on crops and its effects on the honey bees.

Keywords: beekeeping, honey bees, pesticides, transcriptome

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Authors: Zelikha Labbani

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The in vitro isolated microspore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a microspore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the microspore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of microspore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, microspore became a strategy to achieve various objectives particularly in genetic engineering. In this communication we would show the most recent advances in the producing haploid embryos via in vitro isolated microspore culture.

Keywords: in vitro isolated microspore culture, success, haploid cells, bioinformatics, biomedicine

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Authors: Ali Bayram, Burak Uz, Remzi Yiğiter

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

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

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Authors: Yungeng Xie, Cong Liu, Yi Liu, Xuanao Wan

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The study aims to explore microblog users’ emotion expression and sharing behaviors on the Chinese microblog (Weibo). The first theme of study analyzed whether microblog emotions impact readers’ message sharing behaviors, specifically, how the strength of emotion (positive and negative) in microblog messages facilitate/inhibit readers’ sharing behaviors. The second theme compared the differences among the three types of microblog users (i.e., verified enterprise users, verified individual users and unverified users) in terms of their profiles and microblog behaviors. A total of 7114 microblog messages about 24 hot public events in China were sampled from Sina Weibo. The first study results show that strength of negative emotions that microblog messages carry significantly increase the possibility of the message being shared. The second study results indicate that there are significant differences across the three types of users in terms of their emotion expression and its influence on microblog behaviors.

Keywords: emotion expression, information diffusion, microblog, sharing

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Authors: Vandana Singh, Subash Singh

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Introduction: Oral squamous cell carcinoma is the most common malignant tumor of the oral cavity. Normally the death of cell and the growth are active processes and depend not only on external factors but also on the expression of genes like Bcl-2, which activate and inhibit apoptosis. The term Bcl-2 is an acronym for B-cell lymphoma/ leukemia -2 genes. Objectives: An attempt was made to evaluate Bcl-2 oncoprotein expression in patients with oral precancer and cancer and to assess possible correlation between Bcl-2 oncoprotein expression and clinicopathological features of oral precancer and cancer. Material and Methods: This is a selective prospective clinical and immunohistochemical study. Clinicopathological examination is correlated with immunohistochemical findings. The immunolocalization of Bcl-2 protein is performed using the labeled streptavidin biotin (LSAB) method. To visualize the reaction, 3, 3-diaminobenzidine (DAB) is used. Results: Bcl-2 expression was positive in 11 [36.66 %, low Bcl-2 expression 3 (10.00 %), moderate Bcl-2 expression 7 (23.33 %), and high Bcl-2 expression 1 (3.33 %)] oral cancer cases and in 14 [87.50 %, low expression 8 (50 %), moderate expression 6 (37.50 %)] precancer cases. Conclusion: On the basis of the results of our study we conclude that positive Bcl-2 expression may be an indicator of poor prognosis in oral cancer and precancer. Relevance: It has been reported that there is deregulation of Bcl-2 expression during progression from oral epithelial dysplasia to squamous cell carcinoma. It can be used for revealing progression of epithelial dysplasia to malignancy and as a prognostic marker in oral precancer and cancer.

Keywords: BcL-2, immunohistochemistry, oral cancer, oral precancer

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Authors: Tahira Hussain, Ilhom Rahamkulov, Muhammad Aasim, Ugur Pirlak, Emre Aksoy, Mehmet Emin Caliskan, Allah Bakhsh

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RNA interference (RNAi) has proved its usefulness in functional genomic research on insects recently and is considered potential strategy in crop improvement for the control of insect pests. The different insect pests incur significant losses to potato yield worldwide, Colorado Potato Beetle (CPB) being most notorious one. The present study focuses to knock down highly specific 20-hydroxyecdysone hormone-receptor complex interaction by using RNAi approach to silence Ecdysone receptor (EcR) gene of CPB in transgenic potato plants expressing dsRNA of EcR gene. The partial cDNA of Ecdysone receptor gene of CPB was amplified using specific primers in sense and anti-sense orientation and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Lady Olympia, Agria and Granola cultivars of potato were infected with Agrobacterium strain LBA4404 harboring plasmid pRNAi-CPB, pRNAi-GFP (used as control). Neomycin phosphotransferase (nptII) gene was used as a plant selectable marker at a concentration of 100 mg L⁻¹. The primary transformants obtained have shown proper integration of T-DNA in plant genome by standard molecular analysis like polymerase chain reaction (PCR), real-time PCR, Sothern blot. The transgenic plants developed out of these cultivars are being evaluated for their efficacy against larvae as well adults of CPB. The transgenic lines are expected to inhibit expression of EcR protein gene, hindering their molting process, hence leading to increased potato yield.

Keywords: plant mediated RNAi, molecular strategy, ecdysone receptor, insect metamorphosis

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

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The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcuslactis, recombinant, phytase, poultry

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Authors: Mukesh N. Kher, Anju P. Kunjadia, Dev S. Nauriyal, Chaitanya G. Joshi, Navin R. Sheth, Vaibhav D. Bhatt

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In-vivo immunotherapeutic potential on cytokines production and antibacterial activity of a topical herbal gel was evaluated in two breeds of cattle in bovine subclinical mastitis. The response to treatment was evaluated by enumerating somatic cell count (SCC), determining total bacterial count and studying the expression of different cytokines like (interleukin 6, 8, 12, GMCSF, interferon–γ and TNF‑α). The pre‑ and post‑treatment SCC in mastitic quarters did not differ statistically-significantly. However, total bacterial count declined significantly from day 0 onwards in both the breeds. Significant differences (P < 0.01) were observed in all types of cytokines production on day 0, 5, and 21 post last treatments in both the breeds. The comparison of cytokine expression profiles between crossbred and Gir cattle affirmed a significant difference in expression of IL-6 and TNF-α. The topical herbal gel showed immunomodulatory and antimicrobial activities in subclinical mastitis, and therefore the work supports its use as substitute herbal therapy against subclinical mastitis in bovines.

Keywords: antibacterial activity, immunomodulation, herbal gel, subclinical mastitis

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Authors: J. Adeppa, S. Samanta, O. K. Raina

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Fasciolosis is a widespread economically important parasitic infection throughout the world caused by Fasciola hepatica and F. gigantica. In order to identify novel immunogen conferring significant protection against fasciolosis, currently, research has been focused on the defined antigens viz. glutathione S-transferase, fatty acid binding protein, cathepsin-L, fluke hemoglobin, paramyosin, myosin and F. hepatica- Kunitz Type Molecule. Among various antigens, GST which plays a crucial role in detoxification processes, i.e. phase II defense mechanism of this parasite, has a unique position as a novel vaccine candidate and a drug target in the control of this disease. For producing the antigens in large quantities and their purification to complete homogeneity, the recombinant DNA technology has become an important tool to achieve this milestone. RT- PCR was carried out using F. gigantica total RNA as template, and an amplicon of 657 bp GST gene was obtained. TA cloning vector was used for cloning of this gene, and the presence of insert was confirmed by blue-white selection for recombinant colonies. Sequence analysis of the present isolate showed 99.1% sequence homology with the published sequence of the F. gigantica GST gene of cattle origin (accession no. AF112657), with six nucleotide changes at 72, 74, 423, 513, 549 and 627th bp found in the present isolate, causing an overall change of 4 amino acids. The 657 bp GST gene was cloned at BamH1 and HindIII restriction sites of the prokaryotic expression vector pPROEXHTb in frame with six histidine residues and expressed in E. coli DH5α. Recombinant protein was purified from the bacterial lysate under non-denaturing conditions by the process of sonication after lysozyme treatment and subjecting the soluble fraction of the bacterial lysate to Ni-NTA affinity chromatography. Western blotting with rabbit hyper-immune serum showed immuno-reactivity with 25 kDa recombinant GST. Recombinant protein detected F. gigantica experimental as well as field infection in buffaloes by dot-ELISA. However, cross-reactivity studies on Fasciola gigantica GST antigen are needed to evaluate the utility of this protein in the serodiagnosis of fasciolosis.

Keywords: fasciola gigantic, fasciola hepatica, GST, RT- PCR

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Authors: Suwattana Visetnan, Premruethai Supungul, Sureerat Tang, Ikuo Hirono, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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In animals, infection by Gram-negative bacteria and certain viruses activates the Imd signaling pathway wherein the a NF-κB transcription factor, Relish, is a key regulatory protein for the synthesis of antimicrobial proteins. Infection by yellow head virus (YHV) activates the Imd pathway. To investigate the expression of genes involved in YHV infection and under the influence of PmRelish regulation, RNA interference and suppression subtractive hybridization (SSH) are employed. The genes in forward library expressed in shrimp after YHV infection and under the activity of PmRelish were obtained by subtracting the cDNAs from YHV-infected and PmRelish-knockdown shrimp with cDNAs from YHV-infected shrimp. Opposite subtraction gave a reverse library whereby an alternative set of genes under YHV infection and no PmRelish expression was obtained. Sequencing of 252 and 99 cDNA clones from the respective forward and reverse libraries were done and annotated through blast search against the GenBank sequences. Genes involved in defense and homeostasis were abundant in both libraries, 31% and 23% in the forward and reverse libraries, respectively. They were predominantly antimicrobial proteins, proteinases and proteinase inhibitors. The expression of antimicrobial protein genes, ALFPm3, crustinPm1, penaeidin3 and penaeidin5 were tested under PmRelish silencing and Gram-negative bacterium V. harveyi infection. Together with the results previously reported, the expression of penaeidin5 and also penaeidin3 but not ALFPm3 and crustinPm1 were under the regulation of PmRelish in the Imd pathway.

Keywords: relish, yellow head virus, penaeus monodon, antimicrobial proteins

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Authors: Saskya E. Carrera P., Ben Hankamer, Melanie Oey

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The green alga C. reinhardtii provides a platform for the cheap, scalable, and safe production of complex proteins. Despite gene expression in photosynthetic organisms being tightly regulated by light, most expression studies have analysed chloroplast recombinant protein production under constant light. Here the influence of illumination time and intensity on GFP and a GFP-PlyGBS (bacterial-lysin) fusion protein expression was investigated. The expression of both proteins was strongly influenced by the light regime (6-24 hr illumination per day), the light intensity (0-450 E m⁻²s⁻¹) and growth condition (photoautotrophic, mixotrophic and heterotrophic). Heterotrophic conditions resulted in relatively low recombinant protein yields per unit volume, despite high protein yields per cell, due to low growth rates. Mixotrophic conditions exhibited the highest yields at 6 hrs illumination at 200µE m⁻²s⁻¹ and under continuous low light illumination (13-16 mg L⁻¹ GFP and 1.2-1.6 mg L⁻¹ GFP-PlyGBS), as these conditions supported good cell growth and cellular protein yields. A ~23-fold increase in protein accumulation per cell and ~9-fold increase L⁻¹ culture was observed compared to standard constant 24 hr illumination for GFP-PlyGBS. The highest yields under photoautotrophic conditions were obtained under 9 hrs illumination (6 mg L⁻¹ GFP and 2.1 mg L⁻¹ GFP-PlyGBS). This represents a ~4-fold increase in cellular protein accumulation for GFP-PlyGBS. On a volumetric basis the highest yield was at 15 hrs illumination (~2-fold increase L⁻¹ over the constant light for GFP-PlyGBS). Optimising illumination conditions to balance growth and protein expression can thus significantly enhance overall recombinant protein production in C. reinhardtii cultures.

Keywords: chlamydomonas reinhardtii, light, mixotrophic, recombinant protein

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Authors: Hashaam Akhtar

Abstract:

IFNλR1 and IL10R2 collectively construct a heterodimer, which is an acknowledged functional receptor for all type III interferons (IFNs). Expression of IFNλR1 is highly tissue specific, which can help in making type III IFNs a drug of choice as comparable to its analogue, type I IFNs, for treating hepatitis C in the near future. Although, expression of IFNλR1 also varies with the concentration of type I IFNs, but in this study it was shown that the expression of IFNλR1 varies with the protein titers of IFN-α, IFN-λ3 and the newly discovered IFN-λ4. High dosage of IFN-α reduces the expression of IFNλR1 in HepG2 cells, which can affect the antiviral activity of type III IFNs in vivo. We premeditated an experimental strategy to differentiate monocytes into dendritic cells (DCs), type I and type II macrophages in vitro and quantified the expression of the IFNλR1 by qPCR. The exposure of newly discovered IFN-λ4 to macrophages and DCs also raised the expression of its own receptor, which shows that expression of IFN-λ4 protein in hepatitis C patient may augment type I treatment and help ease off viral titers. The results of this study may contribute in some understanding towards the mechanisms involved in the selective expression of IFNLR1 and exceptionalities associated with the receptor.

Keywords: IFNLR1, Interferon Lambda 4 (IFN-λ4), Mononuclear Phagocyte Cells (MPCs), expression

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Authors: Micheal Olaolu Arowolo, Muhammad Azam, Fei He, Mihail Popescu, Dong Xu

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As the quantity of biological articles rises, so does the number of biological route figures. Each route figure shows gene names and relationships. Annotating pathway diagrams manually is time-consuming. Advanced image understanding models could speed up curation, but they must be more precise. There is rich information in biological pathway figures. The first step to performing image understanding of these figures is to recognize gene names automatically. Classical optical character recognition methods have been employed for gene name recognition, but they are not optimized for literature mining data. This study devised a method to recognize an image bounding box of gene name as a photo using deep Siamese neural network models to outperform the existing methods using ResNet, DenseNet and Inception architectures, the results obtained about 84% accuracy.

Keywords: biological pathway, gene identification, object detection, Siamese network

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Authors: Wenjiang Deng, Tian Mou, Yudi Pawitan, Trung Nghia Vu

Abstract:

Estimation of isoform-level gene expression from RNA-seq data depends on simplifying assumptions, such as uniform reads distribution, that are easily violated in real data. Such violations typically lead to biased estimates. Most existing methods provide a bias correction step(s), which is based on biological considerations, such as GC content–and applied in single samples separately. The main problem is that not all biases are known. For example, new technologies such as single-cell RNA-seq (scRNA-seq) may introduce new sources of bias not seen in bulk-cell data. This study introduces a method called XAEM based on a more flexible and robust statistical model. Existing methods are essentially based on a linear model Xβ, where the design matrix X is known and derived based on the simplifying assumptions. In contrast, XAEM considers Xβ as a bilinear model with both X and β unknown. Joint estimation of X and β is made possible by simultaneous analysis of multi-sample RNA-seq data. Compared to existing methods, XAEM automatically performs empirical correction of potentially unknown biases. XAEM implements an alternating expectation-maximization (AEM) algorithm, alternating between estimation of X and β. For speed XAEM utilizes quasi-mapping for read alignment, thus leading to a fast algorithm. Overall XAEM performs favorably compared to other recent advanced methods. For simulated datasets, XAEM obtains higher accuracy for multiple-isoform genes, particularly for paralogs. In a differential-expression analysis of a real scRNA-seq dataset, XAEM achieves substantially greater rediscovery rates in an independent validation set.

Keywords: alternating EM algorithm, bias correction, bilinear model, gene expression, RNA-seq

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Authors: Eun Jung Sohn, Hwan Tae Park

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Autophagy is a cellular response to stress or environment on cell survival. Here, we investigated the role of ectopic expression of miR 200c-3p in autophagy. Ectopic expression of miR 200c-3p increased the expression of IRE1alpha, ATF6 and CHOP by western blot and RT-qPCR. Furthermore, the level of microRNA 200c-3p was enhanced by treatment of TG or overexpression of GRP 78. Also, ectopic expression of miR200c-3p increased the LC3 II expression by western blot and RT-qPCR. Also, we found that western blot assay showed that miR200c-3p inhibitor was blocked the starvation–induced LC3II levels. Furthermore, starvation stress increased the level of miR200c-3p in different kinetics. Ectopic expression of miR200c-3p attenuated LC3II expression in IRE1 siRNA transfected PC3 cells. Here, we first demonstrate that miR200c-3p regulates autophagy via ER stress pathway.

Keywords: Autophagy, ER stress, LC3II, miR200c-3p

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Authors: Waranya Imprasittichai, Patsarawadee Paojinda, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

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Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. In this study, we constructed gene fusions of Plasmodium falciparum OMPDC-OPRT (1,836 bp) in pTrcHisA vector and expressed as an 6xHis-tag bifunctional protein in three Escherichia coli strains (BL21, Rosetta, TOP10) at 18 °C, 25 °C and 37 °C. The recombinant bifunctional protein was partially purified by Ni-Nitrilotriacetic acid-affinity chromatography. Specific activities of OPRT and OMPDC domains in the bifunctional enzyme expressed in E. coli TOP10 cells were approximately 3-4-fold higher than those in BL21 cells. There were no enzymatic activities when the construct vector expressed in Rosetta cells. Maximal expression of the fused gene was observed at 18 °C and the bifunctional enzyme had specific activities of OPRT and OMPDC domains in a ratio of 1:2. These results provide greater yields and better catalytic activities of the bifunctional OMPDC-OPRT enzyme for further purification and kinetic study.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

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Authors: Yen-Ni Teng, Hsing-Yi Chen, Hsien-An Pan, Yung-Ming Lin, Hany A. Omar, Jui-Hsiang Hung

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Leucine-rich repeats and WD repeat domain containing 1 (LRWD1) is highly expressed in the testes of healthy males. On the other hand, LRWD1 is significantly down-regulated in the testicular tissues of patients with severe spermatogenic defects. In our study, the downregulation of LRWD1 expression by shRNA caused a significant reduction of cell growth and mitosis and a noteworthy increase in the cell microtubule atrophy rate. Here, we used EMBOSS CpG plot analysis to explore the promoter region of LRWD1 gene. We found that CpG islands are located between positions -253 to +5 nucleotides upstream from the LRWD1 transcription start site. Luciferase reporter assay revealed that the hypermethylation of the LRWD1 promoter reduced the transcription activity in cells. In addition, quantitative methylation-specific PCR and immunostaining showed that the methylation inhibitor, 5-Aza-2'-deoxycytidine, increased LRWD1 promoter activity, LRWD1 mRNA, protein expression and cell viability. Whereas, the methylation activator, S-adenosylmethionine, caused opposite effects. The overexpression of p53 and Nrf2 in NT2/D1 cells increased LRWD1 promoter activity while 5-fluorodeoxyuridine decreased it. In conclusion, this study highlights evidence that the methylation status of LRWD1 promoter is associated with LRWD1 expression. Since the expression level of LRWD1 plays an important role in spermatogenesis, the methylation status of LRWD1 may serve as a novel molecular diagnostic or therapeutic approach in male's infertility.

Keywords: LRWD1, DNA methylation, p53, Nrf2

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Authors: Yudi Zhao, Ziyun Zhou, Yueting Yao, Shuying Dai, Zhiling Yan, Longyu Yang, Chuanyin Li, Li Shi, Yufeng Yao

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HPV16 E4 gene plays an important role in viral genome amplification and release. Therefore, a variation of the E4 gene nucleic acid sequence may affect the carcinogenicity of HPV16. In order to understand the relationship between the variation of HPV16 E4 gene and cervical cancer, this study was to amplify and sequence the DNA sequences of E4 genes in 118 HPV16-positive cervical cancer patients and 151 HPV16-positive asymptomatic individuals. After obtaining E4 gene sequences, the phylogenetic trees were constructed by the Neighbor-joining method for gene variation analysis. The results showed that: 1) The distribution of HPV16 variants between the case group and the control group differed greatly (P = 0.015)，and the Asian-American（AA）variant was likely to relate to the occurrence of cervical cancer. 2) DNA sequence analysis showed that there were significant differences in the distribution of 8 variants between the case group and the control group (P < 0.05). And 3) In European (EUR) variant, two variations, C3384T (L18L) and A3449G (P39P), were associated with the initiation and development of cervical cancer. The results suggested that the variation of HPV16 E4 gene may be a contributor affecting the occurrence as well as the development of cervical cancer, and different HPV16 variants may have different carcinogenic capability.

Keywords: cervical cancer, HPV16, E4 gene, variations

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Authors: Ramin Mehdiabadi, Neda Menbari, Mohammad Nazir Menbari

Abstract:

As a pressing public health concern, breast cancer stands as the predominant oncological diagnosis and principal cause of cancer-related mortality among women globally, accounting for 11.7% of new cancer incidences and 6.9% of cancer-related deaths. The annual figures indicate that approximately 230,480 women are diagnosed with breast cancer in the United States alone, with 39,520 succumbing to the disease. While developed economies have reported a deceleration in both incidence and mortality rates across various forms of cancer, including breast cancer, emerging and low-income economies manifest a contrary escalation, largely attributable to lifestyle-mediated risk factors such as tobacco usage, physical inactivity, and high caloric intake. Breast cancer is distinctly characterized by molecular heterogeneity, manifesting in specific subtypes delineated by biomarkers—Estrogen Receptors (ER), Progesterone Receptors (PR), and Human Epidermal Growth Factor Receptor 2 (HER2). These subtypes, comprising Luminal A, Luminal B, HER2-enriched, triple-negative/basal-like, and normal-like, necessitate nuanced, subtype-specific therapeutic regimens, thereby challenging the applicability of generalized treatment protocols. Within this molecular complexity, the transcription factor Forkhead Box M1 (FoxM1) has garnered attention as a significant driver of cellular proliferation, tumorigenesis, metastatic progression, and treatment resistance in a spectrum of human malignancies, including breast cancer. Concurrently, microRNAs (miRs), specifically miR-216b-5p, have been identified as post-transcriptional gene expression regulators and potential tumor suppressors. The overarching objective of this academic investigation is to explicate the multifaceted interrelationship between FoxM1 and miR-216b-5p across the disparate molecular subtypes of breast cancer. Employing a methodologically rigorous, interdisciplinary research design that incorporates cutting-edge molecular biology techniques, sophisticated bioinformatics analytics, and exhaustive meta-analyses of extant clinical data, this scholarly endeavor aims to unveil novel biomarker-specific therapeutic pathways. By doing so, this research is positioned to make a seminal contribution to the advancement of personalized, efficacious, and minimally toxic treatment paradigms, thus profoundly impacting the global efforts to ameliorate the burden of breast cancer.

Keywords: breast cancer, fox m1, microRNAs, mir-216b-5p, gene expression

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