Inhibitory Metaplasticity in Juvenile Stressed Rats Restores Associative Memory in Adulthood by Regulating Epigenetic Complex G9a/GLP.

BACKGROUND: Exposure to juvenile stress was found to have long-term effects on the plasticity and quality of associative memory in adulthood, but the underlying mechanisms are still poorly understood. METHODS: Three- to four week-old male Wistar rats were subjected to a 3-day juvenile stress paradigm. Their electrophysiological correlates of memory using the adult hippocampal slice were inspected to detect alterations in long-term potentiation and synaptic tagging and capture model of associativity. These cellular alterations were tied in with the behavioral outcome by subjecting the rats to a step-down inhibitory avoidance paradigm to measure strength in their memory. Given the role of epigenetic response in altering plasticity as a repercussion of juvenile stress, we aimed to chart out the possible epigenetic marker and its regulation in the long-term memory mechanisms using quantitative reverse transcription polymerase chain reaction. RESULTS: We demonstrate that even long after the elimination of actual stressors, an inhibitory metaplastic state is evident, which promotes synaptic competition over synaptic cooperation and decline in latency of associative memory in the behavioral paradigm despite the exposure to novelty. Mechanistically, juvenile stress led to a heightened expression of the epigenetic marker G9a/GLP complex, which is thus far ascribed to transcriptional silencing and goal-directed behavior. CONCLUSIONS: The blockade of the G9a/GLP complex was found to alleviate deficits in long-term plasticity and associative memory during the adulthood of animals exposed to juvenile stress. Our data provide insights on the long-term effects of juvenile stress that involve epigenetic mechanisms, which directly impact long-term plasticity, synaptic tagging and capture, and associative memory.

Intermittent fasting promotes prolonged associative interactions during synaptic tagging/capture by altering the metaplastic properties of the CA1 hippocampal neurons.

Metaplasticity is the inherent property of a neuron or neuronal population to undergo activity-dependent changes in neural function that modulate subsequent synaptic plasticity. Here we studied the effect of intermittent fasting (IF) in governing the interactions of associative plasticity mechanisms in the pyramidal neurons of rat hippocampal area CA1. Late long-term potentiation and its associative mechanisms such as synaptic tagging and capture at an interval of 120 min were evaluated in four groups of animals, AL (Ad libitum), IF12 (daily IF for 12 h), IF16 (daily IF for 16 h) and EOD (every other day IF for 24 h). IF had no visible effect on the early or late plasticity but it manifested a critical role in prolonging the associative interactions between weak and strong synapses at an interval of 120 min in IF16 and EOD animals. However, both IF12 and AL did not show associativity at 120 min. Plasticity genes such as Bdnf and Prkcz, which are well known for their expressions in late plasticity and synaptic tagging and capture, were significantly upregulated in IF16 and EOD in comparison to AL. Specific inhibition of brain derived neurotropic factor (BDNF) prevented the prolonged associativity expressed in EOD. Thus, daily IF for 16 h or more can be considered to enhance the metaplastic properties of synapses by improving their associative interactions that might translate into animprovedmemoryformation.

Correlation of phylogenetic clade diversification and in vitro infectivity differences among Cosmopolitan genotype strains of Chikungunya virus.

Cosmopolitan genotypes of Chikungunya virus caused the large-scale febrile disease outbreaks in the last decade in Asian and African continents. Molecular analyses of these strains had revealed significant genetic diversification and occurrence of novel mosquito-adaptive mutations. In the present study we looked into whether the genetic diversification has implications in the infectivity phenotype. A detailed sequence and phylogenetic analyses of these virus strains of Indian Ocean lineage from Kerala, South India from the years 2008 to 2013 identified three distinct genetic clades (I, II and III), which had presence of clade-specific amino acid changes. The E2 envelope protein of the strains from the years 2012 to 2013 had a K252Q or a novel K252H change. This site is reported to affect mosquito cell infectivity. Most of these strains also had the E2 G82R mutation, a mutation previously identified to increase mammalian cell infectivity, and a novel mutation E2 N72S. Positive selection was identified in four sites in the envelope proteins (E1 K211E, A226V and V291I; E2 K252Q/H). In infectivity analysis, we found that strains from clade III had enhanced cytopathogenicity in HEK293 and Vero cells than by strains representing other two clades. These two strains formed smaller sized plaques and had distinctly higher viral protein expression, infectious virus production and apoptosis induction in HEK293 cells. They had novel mutations R171Q in the nsP1; I539S in nsP2; N409T in nsP3; and N72S in E2. Our study identifies a correlation between phylogenetic clade diversification and differences in mammalian cell infectivity phenotype among Cosmopolitan genotype CHIKV strains.

Phylogenetic study reveals co-circulation of Asian II and Cosmopolitan genotypes of Dengue virus serotype 2 in Nepal during 2013.

The re-emergence of dengue virus in Nepal and the recent widespread disease epidemics of unprecedented magnitude have raised a great public health concern. There are very few reports on Dengue virus (DENV) strains circulating in the country, especially at the molecular phylogenetics level. In this study, clinical samples from an outbreak in Nepal in 2013, which were positive for DENV serotype 2, were characterized by targeted genome sequencing. Envelope protein (E) coding region from fifteen samples were sequenced and compared with DENV-2 sequences of strains from different geographic regions obtained from the GenBank. Compared to the prototype New Guinea C strain, the samples had a total of eleven non-synonymous substitutions in the envelope protein coding region leading to amino acid change at positions 47, 52, 71, 126, 129, 149, 164, 390, 402, 454 and 462. However, none of these sites were found to be positively selected. A major observation was the presence of two distinct genotypes (Cosmopolitan Genotype IVa and Asian II) in the outbreak as seen by the phylogenetic analysis. It gives the first evidence of the introduction of Cosmopolitan Genotype IVa in Nepal. These strains replace the Genotype IVb strains prevalent earlier since 2004. Both genotypes had closer genetic relation to strains from other countries indicating possibility of exotic introduction. The Genotype IVa strain seems to be more adapted in C6/36 mosquito cells as indicated by its marginally increased replication rate than the Asian II strain in in vitro infection kinetics assays. The genotype replacement and co-circulation of two distinct genotypes may have significant consequences in dengue epidemiology and disease dynamics in Nepal in years to come.

Lineage shift in Indian strains of Dengue virus serotype-3 (Genotype III), evidenced by detection of lineage IV strains in clinical cases from Kerala.

BACKGROUND: Local epidemiology of Dengue is defined by the genetic diversity of the circulating Dengue virus (DENV) strains. This important information is not available for the virus strains from most parts of the Indian subcontinent. The present study focused on the genetic diversity of the serotype 3 DENV strains (DENV-3) from India. RESULTS: A total of 22 DENV-3 strains identified by reverse-transcription PCR analysis of serum samples from 709 patients were studied. These samples were collected over a period of 4 years (2008-2011) from dengue fever suspected patients from Kerala, a dengue endemic state in South India. Comparison of a 1740bp nucleotide sequence of the viral Capsid-Pre-membrane-Envelope coding region of our strains and previously reported DENV-3 strains from India, South Asia and South America revealed non-synonymous substitutions that were genotype III-specific as well as sporadic. Evidence of positive selection was detected in the I81 amino acid residue of the envelope protein. Out of the 22 samples, three had I81A and 18 had I81V substitutions. In the phylogenetic analysis by maximum likelihood method the strains from Kerala clustered in two different lineages (lineage III and IV) within genotype III clade of DENV-3 strains. The ten strains that belonged to lineage IV had a signature amino acid substitution T219A in the envelope protein. Interestingly, all these strains were found to be closely related to a Singapore strain GU370053 isolated in 2007. CONCLUSIONS: Our study identifies for the first time the presence of lineage IV strains in the Indian subcontinent. Results indicate the possibility of a recent exotic introduction and also a shift from the existing lineage III strains to lineage IV. Lineage shifts in DENV-3 strains have been attributed to dramatic increase in disease severity in many parts of the world. Hence the present observation could be significant in terms of the clinical severity of future dengue cases in the region.

Molecular characterization of Chikungunya virus isolates from clinical samples and adult Aedes albopictus mosquitoes emerged from larvae from Kerala, South India.

Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by infected Aedes (Ae.) aegypti and Ae.albopictus mosquitoes. In the study, reverse-transcription PCR (RT PCR) and virus isolation detected CHIKV in patient samples and also in adult Ae.albopictus mosquitoes that was derived from larvae collected during a chikungunya (CHIK) outbreak in Kerala in 2009. The CHIKV strains involved in the outbreak were the East, Central and South African (ECSA) genotype that had the E1 A226V mutation. The viral strains from the mosquitoes and CHIK patients from the same area showed a close relationship based on phylogenetic analysis. Genetic characterization by partial sequencing of non-structural protein 2 (nsP2; 378 bp), envelope E1 (505 bp) and E2 (428 bp) identified one critical mutation in the E2 protein coding region of these CHIKV strains. This novel, non-conservative mutation, L210Q, consistently present in both human and mosquito-derived samples studied, was within the region of the E2 protein (amino acids E2 200-220) that determines mosquito cell infectivity in many alpha viruses. Our results show the involvement of Ae. albopictus in this outbreak in Kerala and appearance of CHIKV with novel genetic changes. Detection of virus in adult mosquitoes, emerged in the laboratory from larvae, also points to the possibility of transovarial transmission (TOT) of mutant CHIKV strains in mosquitoes.