Integrated use of bioinformatic resources reveals that co-targeting of histone deacetylases, IKBK and SRC inhibits epithelial-mesenchymal transition in cancer.

With the advent of high-throughput technologies leading to big data generation, increasing number of gene signatures are being published to predict various features of diseases such as prognosis and patient survival. However, to use these signatures for identifying therapeutic targets, use of additional bioinformatic tools is indispensible part of research. Here, we have generated a pipeline comprised of nearly 15 bioinformatic tools and enrichment statistical methods to propose and validate a drug combination strategy from already approved drugs and present our approach using published pan-cancer epithelial-mesenchymal transition (EMT) signatures as a case study. We observed that histone deacetylases were critical targets to tune expression of multiple epithelial versus mesenchymal genes. Moreover, SRC and IKBK were the principal intracellular kinases regulating multiple signaling pathways. To confirm the anti-EMT efficacy of the proposed target combination in silico, we validated expression of targets in mesenchymal versus epithelial subtypes of ovarian cancer. Additionally, we inhibited the pinpointed proteins in vitro using an invasive lung cancer cell line. We found that whereas low-dose mono-therapy failed to limit cell dispersion from collagen spheroids in a microfluidic device as a metric of EMT, the combination fully inhibited dissociation and invasion of cancer cells toward cocultured endothelial cells. Given the approval status and safety profiles of the suggested drugs, the proposed combination set can be considered in clinical trials.

Three-way interaction model with switching mechanism as an effective strategy for tracing functionally-related genes.

Introduction: Identification of functionally-related genes is an important step in understanding biological systems. The most popular strategy to infer functional dependence is to study pairwise correlations between gene expression levels. However, certain functionally-related genes may have a low expression correlation due to their nonlinear interactions. The use of a three-way interaction (3WI) model with switching mechanism (SM) is a relatively new strategy to trace functionally-related genes. The 3WI model traces the dynamic and nonlinear nature of the co-expression relationship of two genes by introducing their link to the expression level of a third gene. Areas covered: In this paper, we reviewed a variety of existing methods for tracing the 3WIs. Furthermore, we provide a comprehensive review of the previous biological studies based on 3WI models. Expert commentary: Comparison of features of these methods indicates that the modified liquid association algorithm has the best efficiency for tracing 3WI between others. The limited number of biological studies based on the 3WI suggests that high computational demand of the available algorithms is a major challenge to apply this approach for analyzing high-throughput omics data.

Therapeutic effects of hydro-alcoholic extract of Achillea wilhelmsii C. Koch on indomethacin-induced gastric ulcer in rats: a proteomic and metabolomic approach.

BACKGROUND: Gastric ulcer is one of the most prevalent diseases worldwide. In Iranian folk medicine, Achillea wilhelmsii (AW) is used as a treatment for gastric ulcer. Previous reports also mentioned Antiulcerogenic properties for this herbal plant. This study investigated the therapeutic effects of Achillea wilhelmsii C. Koch extract on indomethacin-induced gastric lesion in rats, from both proteomic and metabolomic perspectives. METHODS: The rats were divided into 4 groups. Gastric ulceration was induced by a single dose of indomethacin (45 mg/kg) by oral gavage. An amount of 800 mg/kg of AW extract was administered orally. Serum and tissue samples were collected for further investigations. The metabolomic study was performed by 1H-NMR CPMG spectrometry. Proteomic analysis was also executed by using two dimensional gel electrophoresis (2DE) followed by liquid chromatography coupled to tandem mass spectrometry (LC-ESI/MS/MS). Real time PCR was used to confirm some of the genes. RESULTS: The macroscopic and microscopic investigations confirmed the effectiveness of the AW extract. There was a panel of metabolites which showed alteration during gastric lesion development. The levels of some of these metabolite reversed nearly to their control values after the administration of AW extract. There were also changes in the levels of some proteins including Alb, Fabp5, Hspb1, Tagln, Lgals7, Csta and Myl9 which were reversed after AW administration. CONCLUSIONS: Our findings suggested that Achillea wilhelmsii C. Koch extract could be a potential therapy to be used for indomethacin-induced gastric lesion treatment in the future. However, further investigations are needed to confirm the results.

Valproic acid inhibits the protective effects of stromal cells against chemotherapy in breast cancer: Insights from proteomics and systems biology.

Interaction between tumor and stromal cells is beginning to be decoded as a contributor to chemotherapy resistance. Here, we aim to take a system-level approach to explore a mechanism by which stromal cells induce chemoresistance in cancer cells and subsequently identify a drug that can inhibit such interaction. Using a proteomic dataset containing quantitative data on secretome of stromal cells, we performed multivariate analyses and found that bone-marrow mesenchymal stem cells (BM-MSCs) play the most protective role against chemotherapeutics. Pathway enrichment tests showed that secreted cytokines from BM-MSCs activated 4 signaling pathways including Janus kinase-signal transducer and activator of transcription, phosphatidylinositol 3-kinase-protein kinase B, and mitogen-activated protein kinase, transforming growth factor-ß in cancer cells collectively leading to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcription factor activation. Based on the data from integrated Library of Integrated Network-Based Cellular Signatures (iLINCs) program, we found that among different drugs, valproic acid (VA) affected the expression of 34 genes within the identified pathways that are activated by stromal cells. Our in vitro experiments confirmed that VA inhibits NF-kB activation in cancer cells. In addition, analyzing gene expression data in patients taking oral VA showed that this drug decreased expression of antioxidant enzymes culminating in increased oxidative stress in tumor cells. These results suggest that VA confines the protective role of stromal cells by inhibiting the adaptation mechanisms toward oxidative stress which is potentiated by stromal cells. Since VA is an already prescribed drug manifesting anticancer effects, this study provides a mechanistic insight for combination of VA with chemotherapy in the clinical setting.

Chronic ghrelin administration restores hippocampal long-term potentiation and ameliorates memory impairment in rat model of Alzheimer's disease.

Alzheimer's disease (AD), as a common age-related dementia, is a progressive manifestation of cognitive decline following synaptic failure resulted majorly by senile plaques composed of deposits of amyloid beta (Aß). Ghrelin is a multifunctional peptide hormone with receptors present in various brain tissues including hippocampus and has been associated with neuroprotection, neuromodulation, and memory processing. Here, we investigated the neuroprotective and therapeutic effects of intracerebroventricular (icv) ghrelin infusion for 2 weeks on passive avoidance learning (PAL), memory retention, and synaptic plasticity in the hippocampal dentate gyrus (DG) and CA1 of both normal rats and Aß1-42-induced neurotoxicity in AD model. Male Wistar rats were evaluated for their passive memory performance using a shuttle box while some groups had already received Aß1-42 and/or chronic ghrelin. Using field potential recording, the induction of short- and long-term potentiation (STP and LTP) was studied in DG granule cells along with the LTP changes in CA1 pyramidal neurons through stimulation of the medial perforant path (mPP) and Schaffer collaterals (SCs), respectively. Our results demonstrated that chronic ghrelin treatment not only improved memory processing and retrieval in normal rats during the PAL task, but also promoted memory retention and alleviated memory loss by amelioration of Aß1-42-induced synaptic plasticity impairment in AD subjects through augmentation of field excitatory postsynaptic potential (fEPSP) slope that led to LTP restitution in both the mPP-DG and the CA3-CA1 synapses. Meanwhile, STP was not significantly changed, meaning that although ghrelin enhanced postsynaptic excitability in DG, it did not change presynaptic transmitter release significantly. This suggests the involvement of postsynaptic mechanisms in long-term ghrelin-enhanced memory. In conclusion, it can be inferred that chronic ghrelin administration has an auspicious therapeutic value for impaired cognitive performance and memory deficits in AD-like neuropathology.

Display of B. pumilus chitinase on the surface of B. subtilis spore as a potential biopesticide.

BACKGROUND: Chitinases can inhibit the growth of many fungal diseases which are a great threat for global agricultural production. Biological control of pathogens like fungi, is believed to be one of the best ways to eliminate the adverse effects of plant pathogens. To this end, we expressed and displayed a chitinase from Bacillus pumilus (ChiS) on the surface of Bacillus subtilis spores, as a biocontrol agent. RESULT: ChiS enzyme from B. pumilus was expressed on the spores of B. subtilis using CotG as a carrier protein. Immunofluorescence microscopy confirmed the expression of ChiS on the surface of the spores. Enzyme activity assay showed that the surface displayed ChiS was active and was also able to inhibit the growth of Rhizoctonia solani and Trichoderma harzianum fungi. Western blot analysis also indicated that CotG-ChiS is partially processed after display. Molecular dynamics simulation showed that the stability of the heterologous protein was decreased after fusion. CONCLUSION: ChiS was successfully displayed on the surface of Bacillus spores by fusion to the CotG, one of the main spore coat proteins. In-vitro experiments showed that the displayed enzyme was effective in growth inhibition of R. solani and T. harzianum fungi.

Stress increased ghrelin secretion from pancreatic isolated islets in male rats.

It has been demonstrated that plasma ghrelin is likely affected by stress, but little attention has been paid to the effect of stress on ghrelin release from pancreatic islets. This study investigates the effect of stress on ghrelin secretion from pancreatic islets in rats. Male Wistar rats were divided into control and stressed groups. The stressed group was further divided into foot-shock and psychological stress subgroups. Stress was induced by a communication box. After stress exposure, blood sampling was performed to determine the plasma levels of corticosterone, glucose, and ghrelin. Then the animals' pancreatic islets were isolated to assess their ghrelin output at 5.6, 8.3, and 16.7 mM glucose concentrations. Acute exposure to foot-shock and psychological stress both increased plasma corticosterone concentration. Moreover, plasma glucose concentration increased in the foot-shock stress group. Chronic exposure to foot-shock decreased plasma ghrelin concentration, whereas acute exposure had no significant effect. Acute and chronic exposure to foot-shock and psychological stress increased ghrelin secretion from isolated islets in the presence of different glucose concentrations. The results of the present study suggest that ghrelin secretion from isolated islets is not glucose-dependent. However, ghrelin secretion appears to be intensely responsive to both acute and chronic stress.

Chronic administration of ghrelin regulates plasma glucose and normalizes insulin levels following fasting hyperglycemia and hyperinsulinemia.

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. The majority of the previous studies have shown that the short-term ghrelin treatment induces hyperglycemia and hypoinsulinemia in healthy humans and rodents. However, the results obtained from long-term treatment with ghrelin are not clear enough. In this study, we assessed acute (1 day) and chronic (21 days) effects of intraperitoneally administered ghrelin (at different doses of 1, 10 and 20 µg/kg) during a 12-h fasting period in rats using glucose oxidase method and direct sandwich ELISA (the Enzyme-Linked Immunosorbent Assay) and then compared the effects of exogenous ghrelin on blood glucose and insulin levels on day 21 with those on day 1. The results showed that acute ghrelin administration markedly increased fasting plasma glucose at doses of 1 and 10 µg/kg as well as insulin levels at 1 µg/kg in comparison to control values. Ghrelin (at 1 µg/kg) altered plasma glucose but not insulin levels on the 21st day compared to control values. In addition, the comparison of the influence of ghrelin administration on plasma glucose and insulin levels on day 21 with those on the first day revealed that the chronic administration of ghrelin notably decreased plasma glucose and insulin levels relative to the acute ghrelin treatment. These findings indicate that hyperglycemia and hyperinsulinemia caused by the exogenous ghrelin during acute treatment are temporary and prolonged treatment with ghrelin regulates plasma glucose and restores insulin to normal levels, suggesting a possible role for ghrelin in improving insulin resistance.

Berberine chloride improved synaptic plasticity in STZ induced diabetic rats.

Previous studies indicated that diabetes affects synaptic transmission in the hippocampus, leading to impairments of synaptic plasticity and defects in learning and memory. Although berberine treatment ameliorates memory impairment and improves synaptic plasticity in streptozotocin (STZ) induced diabetic rats, it is not clear if the effects are pre- or post-synaptic or both. The aim of this study was to evaluate the effects of berberine chloride on short-term plasticity in inhibitory interneurons in the dentate gyrus of STZ-induced diabetic rats. Experimental groups included: The control, control berberine treated (100 mg/kg), diabetic and diabetic berberine treated (50,100 mg/kg/day for 12 weeks) groups. The paired pulse paradigm was used to stimulate the perforant pathway and field excitatory post-synaptic potentials (fEPSP) were recorded in dentate gyrus (DG). In comparison with control, paired pulse facilitation in the diabetic group was significantly increased (P < 0.01) and this effect prevented by chronic berberine treatment (50,100 mg/kg). However, there were no differences between responses of the control berberine 100 mg/kg treated and diabetes berberine treated (50 and 100 mg/kg) groups as compared to the control group. The present results suggest that the pre-synaptic component of synaptic plasticity in the dentate gyrus is affected under diabetic conditions and that berberine prevents this effect.

RT-DOb, a switch gene for the gene pair {Csf1r, Milr1}, can influence the onset of Alzheimer's disease by regulating communication between mast cell and microglia.

In biology, homeostasis is a central cellular phenomenon that plays a crucial role in survival. The central nervous system (CNS) is controlled by exquisitely sensitive homeostatic mechanisms when facing inflammatory or pathological insults. Mast cells and microglia play a crucial role in CNS homeostasis by eliminating damaged or unnecessary neurons and synapses. Therefore, decoding molecular circuits that regulate CNS homeostasis may lead to more effective therapeutic strategies that specifically target particular subsets for better therapy of Alzheimer's disease (AD). Based on a computational analysis of a microarray dataset related to AD, the H2-Ob gene was previously identified as a potential modulator of the homeostatic balance between mast cells and microglia. Specifically, it plays such a role in the presence of a three-way gene interaction in which the H2-Ob gene acts as a switch in the co-expression relationship of two genes, Csf1r and Milr1. Therefore, the importance of the H2-Ob gene as a potential therapeutic target for AD has led us to experimentally validate this relationship using the quantitative real-time PCR technique. In the experimental investigation, we confirmed that a change in the expression levels of the RT1-DOb gene (the rat ortholog of murine H2-Ob) can switch the co-expression relationship between Csf1r and Milr1. Furthermore, since the RT1-DOb gene is up-regulated in AD, the mentioned triplets might be related to triggering AD.

The effect of ghrelin on antioxidant status in the rat's model of Alzheimer's disease induced by amyloid-beta.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with amyloid-beta (Aß) plaque formation and oxidative stress in the brain. Ghrelin has been proven to exert antioxidant activity and neuroprotection in different neurological diseases. This study is going on to examine the effect of ghrelin on antioxidant status in the rat's model of AD induced by Aß. Cognitive impairment was induced by intra-hippocampal administration of Aß (10 µg) in Wistar rats and ghrelin (80 µg/kg) was administrated intraperitoneal for ten consecutive days. Behavior was assessed with Morris water maze and passive avoidance tests. Malondialdehyde (MDA) level as a marker of lipid peroxidation was assessed using the thiobarbituric acid. Catalase activity was assayed by the decomposition of H2O2. Antioxidant capacity was determined using the FRAP method. Treatment with ghrelin decreased the hippocampus and serum MDA levels in wild-type rodents and prevented an increase in hippocampal and serum MDA levels in animals receiving Aß. There was no significant change in the serum catalase activity between the studied groups. Hippocampus catalase activity was reduced in the Aß group and treatment with ghrelin increased it. The antioxidant capacity of the hippocampus and serum increased in the ghrelin-receiving control group. The hippocampus antioxidant capacity level decreased in the Aß group, and treatment with ghrelin increased it, but there were no significant changes in the serum antioxidant capacity of animals receiving Aß. These results provide evidence that the administration of ghrelin has antioxidant properties and protects against hippocampal lipid peroxidation in a rat model of AD.

Rabies Infection: An Overview of Lyssavirus-Host Protein Interactions.

Viruses are obligatory intracellular parasites that use cell proteins to take the control of the cell functions in order to accomplish their life cycle. Studying the viral-host interactions would increase our knowledge of the viral biology and mechanisms of pathogenesis. Studies on pathogenesis mechanisms of lyssaviruses, which are the causative agents of rabies, have revealed some important host protein partners for viral proteins, especially for most studied species, i.e. Rabies virus. In this review article, the key physical lyssavirus-host protein interactions, their contributions to rabies infection, and their exploitation are discussed to improve the knowledge about rabies pathogenesis.

Rabies virus matrix protein targets host actin cytoskeleton: a protein-protein interaction analysis.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

Nucleus basalis of Meynert modulates signal processing in rat layer 5 somatosensory cortex but leads to memory impairment and tactile discrimination deficits following lesion.

In rodents, exploring through continuous whisking is a process resulted from sensorimotor networking among different layers of somatosensory cortex (SC) such as layer 5 (L5) or barrel field, and regions like the nucleus basalis of Meynert (NBM). NBM is densely packed with cholinergic fibers and its dysfunction leads to diminished acetylcholine release within SC, tactile deficits and Alzheimer's disease (AD)-like memory impairment. Using extracellular single-unit recording, we investigated mechanisms underlying changes in response characteristics of L5b neurons to single or paired deflection of selected principle and adjacent whiskers (PW and AW), following NBM electrical stimulation in normal rats or ibotenic acid-induced NBM lesion leading to potential tactile deficiency and memory loss during passive avoidance learning (PAL) in AD-like neuropathology. Our results indicated that NBM electrical stimulation decreased ON and OFF response magnitude in nearly half of the units upon vibrissal deflection. The larger the response was evoked to whisker deflection before NBM stimulation, the smaller it gets after stimulation. Neuronal spontaneous activity was not changed with NBM stimulation or lesion. Leading to more sublinear response summation and decreased condition-test ratio, NBM lesion decreased ON response magnitude and facilitation, increased AW surround inhibition in paired whisker deflection, increased excitatory and decreased inhibitory receptive fields, weakened information processing during whisking, and resulted in AD-like declined PAL performance. These findings provide further understandings to develop translational approaches in precision therapeutics to target highly specific regions such as NBM or SC, and pathways like cholinergic system involved in tactile and memory deficits in AD.

A Metabolomic Study to Identify Potential Tissue Biomarkers for Indomethacin-Induced Gastric Ulcer in Rats.

BACKGROUND: Gastric Ulcer (GU) is the most prevalent gastrointestinal disorder induced by various factors and Non-Steroid Anti-Inflammatory Drugs (NSAIDs) as one of the most common reasons. Due to the absence of appropriate molecular markers for GU, the aim of this study was to utilize a metabolomics approach in order to find potential metabolite markers for the disease. METHODS: Stomach tissue samples from indomethacin-treated rats and normal controls were used to perform a 1H-NMR metabolomics study. The altered metabolites were identified using random forest multivariate analysis. RESULTS: ROC curves showed that the random forest model had a good predictive performance with AUC of 1 for the test and 0.708 for the training sets. Seventeen differentially expressed metabolites were found between GU and normal tissue sample. These metabolites included trimethylamine, betaine, carnitine, methionine, acetylcho line, choline, N,N-Dimethylglycine, cis-aconitate, tryptophan, spermidine, acetylcarnitine, creatinine, pantothenate, taurine, isoleucine, glucose and kynurenine. CONCLUSION: The results of the study demonstrated that metabolomics approach could serve as a viable method to find potential markers for GU. Surely, further studies are needed for the validation of the results.

Effect of ghrelin on serum metabolites in Alzheimer's disease model rats; a metabolomics studies based on 1H-NMR technique.

OBJECTIVES: Alzheimer's disease (AD) is dysfunction of the central nervous system and as a neurodegenerative disease. The objective of this work is to investigate metabolic profiling in the serum of animal models of AD compared to healthy controls and then to peruse the role of ghrelin as a therapeutic approach for the AD. MATERIALS AND METHODS: Nuclear magnetic resonance (NMR) technique was used for identification of metabolites that are differentially expressed in the serum of a rat model of the AD with or without ghrelin treatment. Using multivariate statistical analysis, models were built and indicated. RESULTS: There were significant differences and high predictive power between AD and ghrelin-treated groups. The area under curve (AUC) of receiver operating characteristic (ROC) curve and Q2 were 0.870 and 0.759, respectively. A biomarker panel consisting of 14 metabolites was identified to discriminate the AD from the control group. Another panel of 12 serum metabolites was used to differentiate AD models from treated models. CONCLUSION: Both panels had good agreements with clinical diagnosis. Analysis of the results displayed that ghrelin improved memory and cognitive abilities. Affected pathways by ghrelin included oxidative stress, and osteoporosis pathways and vascular risk factors.

Lavandula angustifolia Effects on Rat Models of Alzheimer's Disease Through the Investigation of Serum Metabolic Features Using NMR Metabolomics.

BACKGROUND: Alzheimer's Disease (AD) is the most prevalent cause of memory impairment in the elderly population, but the diagnosis and treatment of the disease is still challenging. Lavender aqueous extract has recently been shown to have the potential in clearing Amyloid-beta plaques from AD rat hippocampus. To elucidate the therapeutic mechanisms of lavender, serum metabolic fingerprint of Aß-induced rat Alzheimer's models was investigated through nuclear magnetic resonance spectrometry. METHODS: For the establishment of rat Alzheimer's models, 10 µg of Amyloid beta 1-42 was injected to male Wistar rats. The lavender aqueous extract was injected 20 days after the establishment of the models, once daily for 20 days. Serum samples were collected and metabolite fingerprints were obtained using 500 MHz 1H-NMR spectrometry, following multivariate statistical analyses. The resulted metabolites were then subjected to pathway analysis tools to reveal metabolic pathways affected by the lavender extract treatment. RESULTS: Levels of 10 metabolite markers including alanine, glutamine, serine, isoleucine, valine, carnitine, isobutyrate, pantothenate, glucose and asparagine were reversed nearly to control values after treatment with lavender extract. The results revealed that the most significantly affected pathways during treatment with lavender extract belonged to carbohydrate and amino acid metabolism, including pantothenate and CoA metabolism, glyoxilate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism. CONCLUSION: As lavender extract reversed the direction of changes of some metabolites involved in AD pathogenesis, it was concluded that the extract might play a role in the disease improvement and serve as a potential therapeutic option for the treatment of AD. Moreover, the metabolites which were found in AD rats could serve as a potential marker panel for the disease; however, much further investigation and validation of the results is needed.

Three-way interaction model to trace the mechanisms involved in Alzheimer's disease transgenic mice.

Alzheimer's disease (AD) is the most common cause for dementia in human. Currently, more than 46 million people in the world suffer from AD and it is estimated that by 2050 this number increases to more than 131 million. AD is considered as a complex disease. Therefore, understanding the mechanism of AD is a universal challenge. Nowadays, a huge number of disease-related high-throughput "omics" datasets are freely available. Such datasets contain valuable information about disease-related pathways and their corresponding gene interactions. In the present work, a three-way interaction model is used as a novel approach to understand AD-related mechanisms. This model can trace the dynamic nature of co-expression relationship between two genes by introducing their link to a third gene. Apparently, such relationships cannot be traced by the classical two-way interaction model. Liquid association method was applied to capture the statistically significant triplets which are involved in three-way interaction. Subsequently, gene set enrichment analysis (GSEA) and gene regulatory network (GRN) inference were applied to analyze the biological relevance of the statistically significant triplets. The results of this study suggest that the innate immunity processes are important in AD. Specifically, our results suggest that H2-Ob as the switching gene and the gene pair {Csf1r, Milr1} form a statistically significant and biologically relevant triplet, which may play an important role in AD. We propose that the homeostasis-related link between mast cells and microglia is presumably controlled with H2-Ob expression levels as a switching gene.

Design and expression of a chimeric vaccine candidate for avian necrotic enteritis.

Necrotic enteritis is an economically important disease of poultry mainly caused by Clostridium perfringens The bacteria release multiple toxins of which NetB, alpha toxin and TpeL have been reported to play important roles in pathogenicity and/or severity of the disease. In this study, the sequence of clostridial toxins NetB, alpha toxin and TpeL were analyzed using bioinformatics tools to determine protein domains with high immunogenicity factor. Several chimeric trivalent proteins consisting of the immunogenic regions of the three toxins were designed and evaluated. The separate regions were fused together using rigid linkers. Based on a modeled tertiary structure, a proper combination was selected and expressed in a bacterial host (Escherichia coli) and successfully purified. The expression of the chimeric protein was further verified by western blotting. The ability of the immunized serum in recognizing each individual subunit of the chimeric protein was also examined. Circular dichroism was used to evaluate the predicted secondary structure of the chimeric protein. In vitro potency test demonstrated that the serum from a rabbit immunized with the chimeric protein is able to partially neutralize Alpha toxin, hence the construct can potentially be used as a vaccine against C. perfringens.

The effect of ghrelin on MK-801 induced memory impairment in rats.

Accumulating evidence indicates that the brain-gut peptide ghrelin which is expressed in hippocampus improves memory and learning processes. The MK-801, a noncompetitive NMDA receptor antagonist, has also shown amnesic properties in animal model. The current study was to find out whether intracerebroventricular administration of ghrelin can prevent amnesia induced by MK-801 in rats. A week after the surgery, during which cannuals were implanted in the lateral ventricular, the animals were trained and tested in a step-through type passive avoidance task. Memory retrieval was measured by step-through latency (STL) and total time in dark compartments (TDC). In the first series of experiments, we established a dose-response relationship for ghrelin on the passive avoidance paradigm. In the second set of experiments, animals were divided to two groups. In the first group, MK-801 (0.075, 0.15 and 0.3mg/kg) was injected intraperitoneally (i.p.) immediately after the acquisition session and in the second group MK-801 (same doses) was injected (i.p.) 30 min before the retention session. Analysis of data showed that in both groups, MK-801 impaired learning and memory. In the third set of experiments, administration of ghrelin (200 ng/rat) right after the acquisition session (i.e. before MK-801 injection) improved the MK-801 induced memory impairment, but administration of ghrelin before retrieval session did not affect the MK-801 induced memory impairment. These results show an interaction between ghrelin and glutamatergic system. A novel finding in this study is that ghrelin can prevent amnesia produced by NMDA antagonist in rats when injected in post-training phase.