Transcriptomic profiling of nonneoplastic cortical tissues reveals epileptogenic mechanisms in dysembryoplastic neuroepithelial tumors.

Low-grade dysembryoplastic neuroepithelial tumors (DNTs) are a frequent cause of drug-refractory epilepsy. Molecular mechanisms underlying seizure generation in these tumors are poorly understood. This study was conducted to identify altered genes in nonneoplastic epileptogenic cortical tissues (ECTs) resected from DNT patients during electrocorticography (ECoG)-guided surgery. RNA sequencing (RNAseq) was used to determine the differentially expressed genes (DEGs) in these high-spiking ECTs compared to non-epileptic controls. A total of 477 DEGs (180 upregulated; 297 downregulated) were observed in the ECTs compared to non-epileptic controls. Gene ontology analysis revealed enrichment of genes belonging to the following Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways: (i) glutamatergic synapse; (ii) nitrogen metabolism; (iii) transcriptional misregulation in cancer; and (iv) protein digestion and absorption. The glutamatergic synapse pathway was enriched by DEGs such as GRM4, SLC1A6, GRIN2C, GRM2, GRM5, GRIN3A, and GRIN2B. Enhanced glutamatergic activity was observed in the pyramidal neurons of ECTs, which could be attributed to altered synaptic transmission in these tissues compared to non-epileptic controls. Besides glutamatergic synapse, altered expression of other genes such as GABRB1 (synapse formation), SLIT2 (axonal growth), and PROKR2 (neuron migration) could be linked to epileptogenesis in ECTs. Also, upregulation of GABRA6 gene in ECTs could underlie benzodiazepine resistance in these patients. Neural cell-type-specific gene set enrichment analysis (GSEA) revealed transcriptome of ECTs to be predominantly contributed by microglia and neurons. This study provides first comprehensive gene expression profiling of nonneoplastic ECTs of DNT patients and identifies genes/pathways potentially linked to epileptogenesis.

Role of Altered Expression, Activity and Sub-cellular Distribution of Various Histone Deacetylases (HDACs) in Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis.

Histone deacetylases (HDACs) have been described to have both neurotoxic and neuroprotective roles, and partly, depend on its sub-cellular distribution. HDAC inhibitors have a long history of use in the treatment of various neurological disorders including epilepsy. Key role of HDACs in GABAergic neurotransmission, synaptogenesis, synaptic plasticity and memory formation was demonstrated whereas very less is known about their role in drug-resistant epilepsy pathologies. The present study was aimed to investigate the changes in the expression of HDACs, activity and its sub-cellular distribution in mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) patients. For this study, surgically resected hippocampal tissue specimens of 28 MTLE-HS patients and 20 hippocampus from post-mortem cases were obtained. Real-time PCR was done to analyse the mRNA expression. HDAC activity and the protein levels of HDACs in cytoplasm as well as nucleus were measured spectrophotometrically. Further, sub-cellular localization of HDACs was characterized by immunofluorescence. Significant upregulation of HDAC1, HDAC2, HDAC4, HDAC5, HDAC6, HDAC10 and HDAC11 mRNA were observed in MTLE-HS. Alterations in the mRNA expression of glutamate and gamma-aminobutyric acid (GABA) receptor subunits have been also demonstrated. We observed significant increase of HDAC activity and nuclear level of HDAC1, HDAC2, HDAC5 and HDAC11 in the hippocampal samples obtained from patients with MTLE-HS. Moreover, we found altered cytoplasmic level of HDAC4, HDAC6 and HDAC10 in the hippocampal sample obtained from patients with MTLE-HS. Alterations in the level of HDACs could potentially be part of a dynamic transcription regulation associated with MTLE-HS. Changes in cytoplasmic level of HDAC4, 6 and 10 suggest that cytoplasmic substrates may play a crucial role in the pathophysiology of MTLE-HS. Knowledge regarding expression pattern and sub-cellular distribution of HDACs may help to devise specific HDACi therapy for epilepsy.

Securinine Induces Differentiation of Human Promyelocytic Leukemic HL-60 Cells through JNK-Mediated Signaling Pathway.

Acute myeloid leukemia is characterized by abnormal differentiation of hematopoietic stem cells, leading to the accumulation of immature myeloid cells. Differentiation therapy has been a successful treatment option for acute promyelocytic leukemia but suffers from adverse effects. Therefore, search for novel differentiation-inducing agents with minimal side effects is desirable. Securinine, a naturally-occurring alkaloid, induces differentiation in various leukemic cells and apoptosis in other types of cancers. However, the underlying molecular mechanism(s) remain elusive. Our study aimed to elucidate the possible molecular mechanism(s) and signaling events involved in securinine-induced differentiation of HL-60 cells. Securinine inhibited proliferation in a time- and dose-dependent manner and triggered differentiation. A higher CD14+ population indicated maturation toward monocytic lineage. Securinine caused cell cycle arrest at the G0/G1 phase and enhanced ROS generation. Quantitative gene expression analysis showed significant down-regulation of C/EBP-α, C/EBP-ε, GAΤΑ, and c-myc and up-regulation of the PU.1 gene. The expression of distinct protein kinases Lyn, Chk-2, Yes, FAK, c-Jun, and JNK were enhanced. Use of specific inhibitors of crucial intracellular signaling proteins indicated that JNK and ERK blockade resulted in a significant decline in differentiation. These data thus confirm that securinine induces differentiation through the activation of the JNK-ERK signaling pathway in HL-60 cells.

Altered expression of activating transcription factor 3 in the hippocampus of patients with mesial temporal lobe epilepsy-hippocampal sclerosis (MTLE-HS).

Aim of the study: Activating Transforming factor 3 (ATF3) is a stress induced gene and closely associated with neuro-inflammation while Transforming growth Factor Beta (TGFß) signalling is also reported to be involved in neuro-inflammation and hyper-excitability associated with drug resistant epilepsy. Animal model studies indicate the involvement of ATF3 and TGFß receptors to promote epileptogenesis. Human studies also show that TGFß signalling is activated in MTLE-HS. However, lack of studies on ATF3 and TGFßRI expression in MTLE-HS patients exists. We hypothesize that ATF3 and TGFßRI might be expressed in hippocampi of patients with MTLE-HS and playing role in epileptogenesis.Materials & methods: Protein expression of ATF3 and TGFßRI was performed by western blotting. Localisation of ATF3 was performed by immunohistochemistry and immunoflorescence.Results: Protein expression of ATF3 and TGFßRI was significantly up-regulated in hippocampi of patients as compared to controls. Also ATF3 IR was significantly expressed in hippocampi of patients and ATF3 was expressed predominantly in cytoplasm as compared to nucleus. No correlation was found between ATF3 expression and epilepsy duration and seizure frequency.Conclusions: ATF3 and TGFßRI are both important players in neuro-inflammation and might potentiate epileptogenesis in these patients.

Recombinant outer membrane protein OmpC induces protective immunity against Aeromonashydrophila infection in Labeorohita.

Aeromonashydrophila is an opportunistic pathogen that causes enormous loss to aquaculture industry. The outer membrane proteins of Aeromonas help in bacterium-host interaction, and are considered to be potential vaccine candidates. In the present study, we evaluated immunogenicity and protective efficacy of recombinant OmpC (rOmpC) of A. hydrophila in Indian major carp, Labeorohita. The rOmpC-vaccinated fish produced specific anti-rOmpC antibodies with a significant antibody titer, and the antisera could specifically detect the rOmpC in the cell lysates of Escherichia coli expressing rOmpC and cross-react with different Aeromonas lysates, indicating the suitability of the anti-rOmpC antisera to detect Aeromonas infection. A significant increase was noted in ceruloplasmin level, myeloperoxidase and anti-protease activities in transient and temporal manner the sera of the rOmpC-immunized fish as compared to PBS-control fish. Higher agglutination- and hemolytic activity titers in the anti-rOmpC antisera indicate stimulation of innate immunity. Expression of immune-related genes comprising various acute phase proteins, cytokines and inflammatory response molecules were modulated in the head kidney of rOmpC-immunized L. rohita. While IgM, IL1ß, and TLR-22 were significantly up-regulated at early time points (3 h-72 h), the others showed a transient augmentation at both early and later time points (SOD, lysozymes C and G, NKEF-B, C3, CXCa and TNF-α) in the rOmpC-immunized L. rohita in comparison to PBS-injected controls. These data suggest that the rOmpC-induced immune response is temporally regulated to confer immunity. In vivo challenge of the rOmpC-immunized fish with A. hydrophila showed significantly greater survival when compared to PBS-injected control fish. Thus, our results highlight the immunomodulatory role of rOmpC and demonstrate its protective efficacy in L. rohita, along with the use of anti-rOmpC antisera in detecting Aeromonas infections.

Immunogenic characterization and protective efficacy of recombinant CsgA, major subunit of curli fibers, against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is one of the major pathogens responsible for vibriosis and zoonotic infections in teleosts, marine invertebrates, and also humans through consumption of contaminated or unprocessed seafood. Emergence of resistance against current accessible antibiotics and spread to the food chain and environment necessitate the development of safe and effective subunit vaccine against this bacterium. Many bacteria including V. parahaemolyticus produce extracellular curli fibrils, heteropolymeric filaments of major and minor subunit, which have been implicated in adhesion, biofilm formation, and virulence. Adhesins are the primary contact points with the host which help in establishing infection and colonization. CsgA, an adhesin, is the major structural component of the curli fiber that forms homopolymers of several hundred units. Due to their exposure on the cell surface, the curli fibers are recognized by the host's immune system, would generate high immune response, and therefore can serve as effective vaccine candidate. In the present study, we describe characterization of the csgA gene, and preparation of recombinant soluble CsgA of V. parahaemolyticus (rVpCsgA), and evaluation of its vaccine potential. Immunization of BALB/c mice with the rVpCsgA mounted a strong immune response. Cellular immune assays such as antibody isotyping, in vitro splenocyte proliferation analysis, and cytokine profiling revealed mixed T-helper cell immune response. The anti-rVpCsgA antiserum was agglutination positive and specifically cross-reacted with the curli CsgA present on the outer membrane of V. parahaemolyticus cells, thus demonstrating its neutralization potential. One hundred percent survival of the immunized mice upon challenge with the lethal dosage of the bacterium established that the rVpCsgA could serve as an effective vaccine against the bacterium. KEY POINTS: • Recombinant histidine-tagged CsgA of V. parahaemolyticus, rVpCsgA, was purified. • The rVpCsgA immunization produced mixed immune response and agglutinating antibodies. • Immunization with the rVpCsgA protected mice against V. parahaemolyticus challenge.

α7 nicotinic receptors contributes to glutamatergic activity in the hippocampus of patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS).

Hyperglutamatergic activity in the hippocampus is a major feature of patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Here we investigated whether tonic α7 nicotinic receptor (nAChR) activity could contribute to enhanced glutamatergic activity in the hippocampus of patients with MTLE-HS. Results showed that frequency and amplitude of glutamatergic events recorded from pyramidal neurons in the hippocampal samples obtained from patients with MTLE-HS were altered by α7 nAChR antagonist, methyllycaconitine, suggesting α7 nAChRs may influence hyperexcitability in MTLE-HS.

Structural-functional characterization of recombinant Apolipoprotein A-I fromLabeo rohitademonstrates heat-resistant antimicrobial activity.

Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates. Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed in Escherichia coli BL21(DE3) pLysS expression host as a soluble protein under optimized conditions. The yield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conserved characteristic across mammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namely Aeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tarda in a dose-dependent manner. Minimum bactericidal concentration for A. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 µg/ml or 0.81 µM whereas for A. hydrophila, E. tarda, V. parahaemolyticus and V. harveyi, it was determined to be 100 µg/ml or 3.23 µM. These data strongly suggest that recombinant ApoA-I from Labeo rohita could play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.

Modulation of immune response and protective efficacy of recombinant outer-membrane protein F (rOmpF) of Aeromonas hydrophila in Labeo rohita.

The outer-membrane proteins (OMPs) of Aeromonas hydrophila, an imperative fish pathogen accountable for massive economic losses to aquaculture industry, are found to be immunogenic and considered as potential vaccine candidates. In spite of development in the formulation of vaccine candidates against Aeromonas infection, no commercial preparation has been done so far; in addition, the molecular mechanisms of immunoprotection induced by various vaccine formulations in Indian major carp, Labeo rohita, are little known. The present study was undertaken to evaluate the modulation of immunity and expression of immune-related genes post-rOmpF (recombinant outer-membrane protein of A. hydrophila, a novel vaccine candidate) immunization and protective efficacy after A. hydrophila challenge. The rOmpF-immunized fish showed a variable expression of the immune-related genes, viz. toll-like receptor 22 (TLR), complement component 3 (C3), chemokine (CXCa), tumor necrosis factor-α (TNFα), interleukin 1ß (IL-1ß), manganese superoxide dismutase (MnSOD) and natural killer enhancing factor (NKEF) in the head kidney tissues, when compared to the control group at different time intervals post-vaccination. A significant increase in serum hemolysin titer, ceruloplasmin level and myeloperoxidase activity was observed on day 140 post immunization. Also, bacterial agglutination titer and antiprotease activity were significantly increased on day 42 post immunization. No significant change was observed in lysozyme activity. Challenge studies with live A. hydrophila on day 140 post-immunization of L. rohita significantly increased the relative percentage survival (∼44%) in the vaccinated group. The results suggest that the rOmpF could be used as a potential vaccine candidate to combat A. hydrophila infection in fish.

Synthesis of peptide based epsilon toxin vaccine by covalent anchoring to tetanus toxoid.

The epsilon toxin (Etx) produced by Clostridium perfringens type B and D causes severe enterotoxaemia associated with a general edema and neurological alterations, leading to subsequent death and is listed as one of the most lethal toxins. Currently employed vaccines against C. perfringens epsilon toxin include toxoid based vaccines. Use of peptide vaccines has become an interesting approach for vaccination after the successful licensing of peptide vaccines against Haemophilus influenza, Neisseria meningitides and Streptococcus pneumonia that have demonstrated the potential and effectiveness of these vaccines. Therefore, the present study was undertaken to develop a peptide based vaccine against epsilon toxin. Peptides were selected on the basis of epitope mapping by making 35 overlapping peptides of 15 amino acid residues in length specific to the primary amino acid sequence of the toxin, with a 7 amino acid residues overlaps between sequential peptides. Chemically synthesized peptides that were recognised by the antibody against the full length epsilon toxin were further assessed for vaccine potential. The selected peptides were chemically conjugated to partially reduced tetanus toxoid (TT) using of N-succinimidyl-3(2-pyridyldithio) propionate. Immunization of BALB/c mice with TT-peptide conjugates by sub-cutaneous route induced sustained high level mixed immune response as analyzed by antibody isotyping. Immunoblot analysis and ELISA clearly indicated generation of Etx-specific antibodies. Further, neutralization studies with the antisera generated against the TT-conjugated peptide(s) demonstrated that the antisera were able to neutralize the lethal dose of epsilon toxin in vitro demonstrating its potential as a promising vaccine candidate against enterotoxaemia.

Clostridium perfringens beta toxin DNA prime-protein boost elicits enhanced protective immune response in mice.

Clostridium perfringens beta toxin (CPB) is the primary pathogenic factor responsible for necrotic enteritis in sheep, cattle and humans. Owing to rapid progression of the disease, vaccination is the only possible recourse to avoid high mortality in animal farms and huge economic losses. The present study reports evaluation of a cpb gene-based DNA vaccine encoding the beta toxin of C. perfringens with homologous as well as heterologous booster strategy. Immunization strategy employing heterologous booster with heat-inactivated rCPB mounted stronger immune response when compared to that generated by homologous booster. Antibody isotyping and cytokine ELISA demonstrated the immune response to be Th1-biased mixed immune response. While moderate protection of immunized BALB/c and C57BL/6 mice against rCPB challenge was observed with homologous booster strategy, heterologous booster strategy led to complete protection. Thus, beta toxin-based DNA vaccine using the heterologous prime-boosting strategy was able to generate better immune response and conferred greater degree of protection against high of dose rCPB challenge than homologous booster regimen, making it an effective vaccination approach against C. perfringens beta toxin.

Synaptic roles of cyclin-dependent kinase 5 & its implications in epilepsy.

There is an urgent need to understand the molecular mechanisms underlying epilepsy to find novel prognostic/diagnostic biomarkers to prevent epilepsy patients at risk. Cyclin-dependent kinase 5 (CDK5) is involved in multiple neuronal functions and plays a crucial role in maintaining homeostatic synaptic plasticity by regulating intracellular signalling cascades at synapses. CDK5 deregulation is shown to be associated with various neurodegenerative diseases such as Alzheimer's disease. The association between chronic loss of CDK5 and seizures has been reported in animal models of epilepsy. Genetic expression of CDK5 at transcriptome level has been shown to be abnormal in intractable epilepsy. In this review various possible mechanisms by which deregulated CDK5 may alter synaptic transmission and possibly lead to epileptogenesis have been discussed. Further, CDK5 has been proposed as a potential biomarker as well as a pharmacological target for developing treatments for epilepsy.

Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression.

Packaging specific exogenous active proteins and DNAs together within a single viral-nanocontainer is challenging. The bacteriophage T4 capsid (100 × 70 nm) is well suited for this purpose, because it can hold a single long DNA or multiple short pieces of DNA up to 170 kb packed together with more than 1,000 protein molecules. Any linear DNA can be packaged in vitro into purified procapsids. The capsid-targeting sequence (CTS) directs virtually any protein into the procapsid. Procapsids are assembled with specific CTS-directed exogenous proteins that are encapsidated before the DNA. The capsid also can display on its surface high-affinity eukaryotic cell-binding peptides or proteins that are in fusion with small outer capsid and head outer capsid surface-decoration proteins that can be added in vivo or in vitro. In this study, we demonstrate that the site-specific recombinase cyclic recombination (Cre) targeted into the procapsid is enzymatically active within the procapsid and recircularizes linear plasmid DNA containing two terminal loxP recognition sites when packaged in vitro. mCherry expression driven by a cytomegalovirus promoter in the capsid containing Cre-circularized DNA is enhanced over linear DNA, as shown in recipient eukaryotic cells. The efficient and specific packaging into capsids and the unpackaging of both DNA and protein with release of the enzymatically altered protein-DNA complexes from the nanoparticles into cells have potential in numerous downstream drug and gene therapeutic applications.

RNA-seq analysis of hippocampal tissues reveals novel candidate genes for drug refractory epilepsy in patients with MTLE-HS.

Array-based profiling studies have shown implication of aberrant gene expression patterns in epileptogenesis. We have performed transcriptome analysis of hippocampal tissues resected from patients with MTLE-HS using RNAseq approach. Healthy tissues from tumour margins obtained during tumour surgeries were used as non-epileptic controls. RNA sequencing was performed using standard protocols on Illumina HiSeq 2500 platform. Differential gene expression analysis of the RNAseq data revealed 56 significantly regulated genes in MTLE patients. Gene cluster analysis identified 3 important hubs of genes mostly linked to, neuroinflammation and innate immunity, synaptic transmission and neuronal network modulation which are supportive of intrinsic severity hypothesis of pharmacoresistance. This study identified various genes like FN1 which is central in our analysis, NEUROD6, RELN, TGFßR2, NLRP1, SCRT1, CSNK2B, SCN1B, CABP1, KIF5A and antisense RNAs like AQP4-AS1 and KIRREL3-AS2 providing important insight into the understanding of the pathophysiology or genomic basis of drug refractory epilepsy due to MTS.

Recent advances in Epilepsy Research in India.

There are more than 10 million persons with epilepsy (PWE) in India. Despite availability of antiepileptic drugs (AEDs), there is a large treatment gap varying from 50 to 70% among PWE. For treatable epilepsy, this gap can be attributed to poor education, poverty, cultural beliefs, stigma, and poor healthcare infrastructure; whereas for chronic epilepsy, this gap can be attributed to lack of proper diagnosis and treatment. To prevent, treat, and cure epilepsy, researchers worldwide have made exciting advances across all areas of epilepsy research. Studies carried out in India have also shown substantial progress; however, most of them are focused on the epidemiological aspects of epilepsy, genetic associations, identification, and validation of new AEDs in animal models of epilepsy.Very few studies are reported on understanding the process of epileptogenesis, a dynamic process by which neurons begin to display abnormal firing patterns that cause epileptic seizures. Animal epilepsy models can be used for in depth studies; however, studies conducted on resected brain tissues from epilepsy patients are clinically relevant. Finally, more funding support from government and collaborations among basic research institutes, medical institutes, as well as industries is required to raise the standards of epilepsy research in India.This review focuses on the evaluation of the current status of epilepsy research in India and the need to identify potential anti-epileptogenic interventions.

Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris.

Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal ß strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50 of ∼3.7 µg/ml and trypsin inhibitor activity with an IC50 of 4.2 µM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source.

Identification and immunogenic potential of B cell epitopes of outer membrane protein OmpF of Aeromonas hydrophila in translational fusion with a carrier protein.

Aeromonas hydrophila, a ubiquitous and virulent bacterial pathogen, affects a variety of fishes, including Labeo rohita. Existing treatment strategies comprise antibiotic therapies and attenuated bacterial strain-based vaccines. No functional subunit vaccine has been available until now. Given their key role in determining pathogenicity, outer membrane proteins have been successfully explored as potential vaccine candidates. We have devised a direct strategy for eliminating non-specific responses by selectively aiming the immune response against specific immunodominant epitopes of the outer membrane protein F (OmpF) of A. hydrophila (AhOmpF). Five putative epitopes of AhOmpF predicted in silico were genetically conjugated with heat labile enterotoxin chain B of E. coli (LTB). Recombinant fusion proteins expressed in E. coli were purified from solubilized inclusion bodies and refolded. The fusion protein retained GM1 ganglioside receptor binding activity of LTB, indicating proper folding. Four of the five fusion proteins were found to be highly immunogenic. Of the four proteins, antisera against the fusion protein (anti-rEpiF1) harboring 66-80 amino acid residues of the OmpF gave maximum cross-reactivity with the targeted rOmpF in enzyme-linked immunosorbent assay (ELISA) and was able to recognize both fusion partners-rOmpF and rLTB-in Western blot. Antibody isotyping of the antisera and cytokine array analysis of the culture supernatants of splenocytes from sensitized mice manifested a mixed Th1/Th2 immune response with a bias toward Th2. Anti-rEpiF1 antibodies were able to bind to the cell membrane of live A. hydrophila cells and agglutinate them. Our results thus suggest that the OmpF epitope (66-80) in fusion with a carrier protein is a promising vaccine candidate against A. hydrophila.

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation.

Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.

Presurgical epileptogenic network analysis: A way to enhance epilepsy surgery outcome.

Accurate localization of the "epileptogenic zone (EZ)" is an important issue in epilepsy surgery. The EZ is not discrete and focal; in fact, the epileptogenic networks can spread ictal activity to different regions of the brain. Changes in network characteristics and functional connectivity are shown to be associated with epilepsy. Seizures are thought to represent a hyper-synchronous state and presumable changes in synchronization between different brain regions underlie the mechanisms of seizure spread. Although presurgical evaluation of the epileptogenic network analysis can be carried out using existing investigative techniques like electroencephalogram (EEG), video-EEG, magnetic resonance imaging, single-photon emission computed tomography, and magnetoencephalography, advanced imaging techniques such as optical intrinsic spectroscopy, auto-fluorescence imaging, voltage sensitive dye imaging, and calcium imaging have the advantage of better spatiotemporal resolution over a large area of cortex. Understanding the wide-scale dynamic networks by analyzing the changes in the synchronization patterns using advanced imaging techniques will be instrumental in the presurgical analysis of the epileptogenic network and better localization of the EZs in the future.

Multivariate sequence analysis reveals additional function impacting residues in the SDR superfamily.

The "extended" type of short chain dehydrogenases/reductases (SDR), share a remarkable similarity in their tertiary structures inspite of being highly divergent in their functions and sequences. We have carried out principal component analysis (PCA) on structurally equivalent residue positions of 10 SDR families using information theoretic measures like Jensen-Shannon divergence and average shannon entropy as variables. The results classify residue positions in the SDR fold into six groups, one of which is characterized by low Shannon entropies but high Jensen-Shannon divergence against the reference family SDR1E, suggesting that these positions are responsible for the specific functional identities of individual SDR families, distinguishing them from the reference family SDR1E. Site directed mutagenesis of three residues from this group in the enzyme UDP-Galactose 4-epimerase belonging to SDR1E shows that the mutants promote the formation of NADH containing abortive complexes. Finally, molecular dynamics simulations have been used to suggest a mechanism by which the mutants interfere with the re-oxidation of NADH leading to the formation of abortive complexes.