Development of RNAi-based genetic tools to silence MKP3 expression in a cell-specific manner

Drug addiction is believed to occur, in part, as a result of maladaptive changes in dopaminergic pathways. The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway is implicated in altering dopaminergic signaling. Previous work demonstrated that MAP kinase phosphatase-3 (MKP3), a negative regulator of the ERK1/2 pathway, modulates dopamine release by altering dopamine transporter surface expression and voltage-gated calcium channel expression. Most pharmacological and genetic tools lack the ability to modulate intracellular signaling pathways in vivo in a cell-specific manner. Development of such cell-specific genetic tools can be used to study the role of long term plasticity changes in pathologies such as drug addiction. The goal of this project was to develop a cell-specific RNAi-based genetic tool to target and alter MKP3 expression in vivo. Cre recombinase-dependent expression of a microRNA-30-based shRNA vector is used to silence MKP3 expression. Cre recombinase reorients a transcript of interest and only expresses it in cells containing Cre. An inverted shRNA construct is flanked by incompatible lox P sites, and Cre recombinase reverses the orientation of the construct to enable cell-specific MKP3 silencing in vivo. This study tested 4 shRNAs that target different regions of the MKP3 reading frame to determine which shRNA induces the most effective MKP3 silencing in vitro. shRNAs were first cloned into DNA vectors that contain incompatible lox P sites and a promoter needed for expression of the shRNA. Proper cloning of shRNAs was verified with agarose gel analysis and DNA sequencing. After optimizing transfection conditions, in vitro studies showed that only 2 shRNAs significantly silenced MKP3 expression; only 1 of those shRNAs showed good red fluorescent protein (RFP) expression. Since these shRNAs were oriented in the forward direction, adding Cre to the shRNA samples reversed their orientation and prevented their expression, which was observed through reduced RFP expression and reversal of the MKP3 silencing effects seen without Cre. Thus both MKP3 silencing and RFP expression are Cre dependent. Future in vivo studies will assess MKP3’s role in cocaine addiction by using the most effective shRNA to silence MKP3 expression only in dopaminergic neurons that contain Cre.M.S., Drug Discover and Development -- Drexel University, 201

Development of RNAi-based genetic tools to silence MKP3 expression in a cell-specific manner

Drug addiction is believed to occur, in part, as a result of maladaptive changes in dopaminergic pathways. The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway is implicated in altering dopaminergic signaling. Previous work demonstrated that MAP kinase phosphatase-3 (MKP3), a negative regulator of the ERK1/2 pathway, modulates dopamine release by altering dopamine transporter surface expression and voltage-gated calcium channel expression. Most pharmacological and genetic tools lack the ability to modulate intracellular signaling pathways in vivo in a cell-specific manner. Development of such cell-specific genetic tools can be used to study the role of long term plasticity changes in pathologies such as drug addiction. The goal of this project was to develop a cell-specific RNAi-based genetic tool to target and alter MKP3 expression in vivo. Cre recombinase-dependent expression of a microRNA-30-based shRNA vector is used to silence MKP3 expression. Cre recombinase reorients a transcript of interest and only expresses it in cells containing Cre. An inverted shRNA construct is flanked by incompatible lox P sites, and Cre recombinase reverses the orientation of the construct to enable cell-specific MKP3 silencing in vivo. This study tested 4 shRNAs that target different regions of the MKP3 reading frame to determine which shRNA induces the most effective MKP3 silencing in vitro. shRNAs were first cloned into DNA vectors that contain incompatible lox P sites and a promoter needed for expression of the shRNA. Proper cloning of shRNAs was verified with agarose gel analysis and DNA sequencing. After optimizing transfection conditions, in vitro studies showed that only 2 shRNAs significantly silenced MKP3 expression; only 1 of those shRNAs showed good red fluorescent protein (RFP) expression. Since these shRNAs were oriented in the forward direction, adding Cre to the shRNA samples reversed their orientation and prevented their expression, which was observed through reduced RFP expression and reversal of the MKP3 silencing effects seen without Cre. Thus both MKP3 silencing and RFP expression are Cre dependent. Future in vivo studies will assess MKP3’s role in cocaine addiction by using the most effective shRNA to silence MKP3 expression only in dopaminergic neurons that contain Cre.M.S., Drug Discover and Development -- Drexel University, 201

Entrepreneurial Behaviour of the Agriculture Students-A review

The present study aimed to investigate the factors that influence the entrepreneurial behavior of college students. The study identified various personal and situational factors that may affect entrepreneurial behavior. The results showed that personality traits, such as openness, extraversion, and conscientiousness, had a significant positive impact on entrepreneurial behavior. Specific motivational traits, such as entrepreneurial self-efficacy, internal locus of control, and risk-taking propensity, were also significant predictors of entrepreneurial behavior. Situational factors, such as entrepreneurship education, were found to have a significant positive impact on entrepreneurial behavior, with entrepreneurial self-efficacy playing a key mediating role in this relationship. Attitudes towards entrepreneurship were found to be a significant driver of entrepreneurial intention, with perceived desirability and feasibility, as well as perceived individual and collective efficacy, also significant predictors of entrepreneurial intention. Sustainable entrepreneurial intention was found to be influenced by attitude towards the behavior variable, with subjective norms playing an indirect role in mediating this effect. Overall, the study suggests that personal traits, such as personality and motivational factors, as well as situational factors, such as education and attitudes towards entrepreneurship, are significant predictors of entrepreneurial behavior. These findings have important implications for educators and policymakers who seek to promote entrepreneurial behavior among college students. Future research should continue to explore the complex relationships between personal and situational factors and entrepreneurial behavior to further enhance our understanding of this important phenomenon. &nbsp

Meningoencephalitis in farmed monosex Nile tilapia (Oreochromis niloticus L.) caused by Streptococcus agalactiae

Aquaculture of tilapia is a new research venture in India. With intensification in farming practices, tilapia are increasingly susceptible to bacterial infections. This article describes the isolation and identification of pathogenic bacteria from cultured monosex Nile tilapia, Oreochromis niloticus (L.), that experienced moderate to severe mortalities in West Bengal, India between September and August 2014 and histopathological alterations in various organs. Gram-positive diplococci, identified as Streptococcus agalactiae with Streptococcus identification kits and 16S rDNA sequencing analysis, were isolated from the brain, operculum, and kidney. Other bacteria from the kidney were identified as Aeromonas sobria, A. caviae, Klebsiella pneumoniae ssp. pneumoniae, Escherichia coli, and Enterobacter cloacae. Staphylococcus epidermis was isolated from opercular hemorrhages. Histological sections of the infected tilapia brain revealed meningoencephalitis and granulomatous lesions. Sections from other organs indicated congestion, hemorrhagic and hyperplastic cells, necrosis, vacuolation, hemosiderin deposition, hypertrophic nuclei, melanomacrophage aggregation, and ruptured veins. This report is the first description of S. agalactiae as a primary pathogen causing meningoencephalitis in cultured tilapia in India

Molecular phylogeny of Myxobolus orissae (Myxosporea: Myxobolidae) infecting the gill lamellae of mrigal carp Cirrhinus mrigala (Actinopterygii: Cyprinidae)

ABSTRACT Myxosporeans are best known for the diseases they cause in commercially important fish species. Identification of myxosporeans at the species-level is mainly based on conventional methods. The 18S rRNA gene sequence of morphologically identified Myxobolus orissae infecting the gill lamellae of mrigal carp Cirrhinus mrigala was characterized in the present study. The plasmodia of M. orissae were small, elongated and white to pale in colour. Phylogenetically, the 18S rDNA nucleotide sequence of M. orissae was clustered with other gill-infecting Myxobolus spp. of cyprinids. The species closely related to M. orissae was M. koi (FJ841887) infecting the gill lamellae of Cyprinus carpio with 96% similarity. The carp fin-infecting Thelohanellus caudatus (KC865607) from India exhibited only 78% DNA sequence similarity with M. orissae. Low level of M. orissae infection on gill caused thickening of epithelial cells surrounding the plasmodium. Under stressful conditions, it is likely that such infection can easily spread in confined fish and may cause serious disease outbreaks and economical losses