Anti-inflammatory and antioxidant effects of muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus

Recently we found that acute treatment with Oxotremorine (Oxo), a non-selective mAChRs agonist, up-regulates heat shock proteins and activates their transcription factor heat shock factor 1 in the rat hippocampus. Here we aimed to investigate: a) if acute treatment with Oxo may regulate pro-inflammatory or anti-inflammatory cytokines and oxidative stress in the rat hippocampus; b) if chronic restraint stress (CRS) induces inflammatory or oxidative alterations in the hippocampus and whether such alterations may be affected by chronic treatment with Oxo. In the acute experiment, rats were injected with single dose of Oxo (0.4 mg/kg) and sacrificed at 24 h, 48 h and 72 h. In the CRS experiment, the rats were exposed for 21 days to the CRS and then were treated with Oxo (0.2 mg/kg) for further 10 days. The acute Oxo treatment showed an ability to significantly reduce reactive oxygen species (ROS), singlet oxygen (1O2), pro-inflammatory cytokines levels (IL-1β and IL-6) and phosphorylated NF-κB-p65. Acute Oxo treatment also increased superoxide dismutase (SOD)-2 protein levels and stimulated SOD activity. No differences were detected in the anti-inflammatory cytokine levels, including IL-10 and TGF-β1. In the group of rats exposed to the CRS were found increased hippocampal IL-1β and IL-6 levels, together with a reduction of SOD activity level. These changes produced by CRS were counteracted by chronic Oxo treatment. In contrast, the upregulation of ROS and 1O2 levels in the CRS group was not counteracted by chronic Oxo treatment. The results revealed a hippocampal anti-inflammatory and antioxidant effect of Oxo treatment in both basal conditions and anti-inflammatory in the CRS rat model

An increased burden of rare exonic variants in NRXN1 microdeletion carriers is likely to enhance the penetrance for autism spectrum disorder.

Autism spectrum disorder (ASD) is characterized by a complex polygenic background, but with the unique feature of a subset of cases (~15%-30%) presenting a rare large-effect variant. However, clinical interpretation in these cases is often complicated by incomplete penetrance, variable expressivity and different neurodevelopmental trajectories. NRXN1 intragenic deletions represent the prototype of such ASD-associated susceptibility variants. From chromosomal microarrays analysis of 104 ASD individuals, we identified an inherited NRXN1 deletion in a trio family. We carried out whole-exome sequencing and deep sequencing of mitochondrial DNA (mtDNA) in this family, to evaluate the burden of rare variants which may contribute to the phenotypic outcome in NRXN1 deletion carriers. We identified an increased burden of exonic rare variants in the ASD child compared to the unaffected NRXN1 deletion-transmitting mother, which remains significant if we restrict the analysis to potentially deleterious rare variants only (P = 6.07 7 10-5 ). We also detected significant interaction enrichment among genes with damaging variants in the proband, suggesting that additional rare variants in interacting genes collectively contribute to cross the liability threshold for ASD. Finally, the proband's mtDNA presented five low-level heteroplasmic mtDNA variants that were absent in the mother, and two maternally inherited variants with increased heteroplasmic load. This study underlines the importance of a comprehensive assessment of the genomic background in carriers of large-effect variants, as penetrance modulation by additional interacting rare variants to might represent a widespread mechanism in neurodevelopmental disorders

Molecular Epidemiology of HIV-1 Transmission in a Cohort of HIV-1 Concordant Heterosexual Couples from Dakar, Senegal

BACKGROUND: A large number of HIV-1 infections in Africa occur in married couples. The predominant direction of intracouple transmission and the principal external origins of infection remain important issues of debate. METHODS: We investigated HIV-1 transmission in 46 HIV-1 concordant positive couples from Dakar, Senegal. Intracouple transmission was confirmed by maximum-likelihood phylogenetic analysis and pairwise distance comparisons of HIV-1 env gp41 sequences from both partners. Standardized interview data were used to deduce the direction as well as the external sources of the intracouple transmissions. RESULTS: Conservative molecular analyses showed linked viruses in 34 (74%) couples, unlinked viruses in 6 (13%) couples, and indeterminate results for 6 (13%) couples. The interview data corresponded completely with the molecular analyses: all linked couples reported internal transmission and all unlinked couples reported external sources of infection. The majority of linked couples (93%) reported the husband as internal source of infection. These husbands most frequently (82%) reported an occasional sexual relationship as external source of infection. Pairwise comparisons of the CD4 count, antiretroviral therapy status, and the proportion of gp41 ambiguous base pairs within transmission pairs correlated with the reported order of infection events. CONCLUSIONS: In this suburban Senegalese population, a majority of HIV-1 concordant couples showed linked HIV-1 transmission with the husband as likely index partner. Our data emphasize the risk of married women for acquiring HIV-1 as a result of the occasional sexual relationships of their husbands

Improved nucleotide selectivity and termination of 3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates

We describe a novel 3′-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2′-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I–dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3′-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies

Chick Embryo Partial Ischemia Model: A New Approach to Study Ischemia Ex Vivo

Background: Ischemia is a pathophysiological condition due to blockade in blood supply to a specific tissue thus damaging the physiological activity of the tissue. Different in vivo models are presently available to study ischemia in heart and other tissues. However, no ex vivo ischemia model has been available to date for routine ischemia research and for faster screening of anti-ischemia drugs. In the present study, we took the opportunity to develop an ex vivo model of partial ischemia using the vascular bed of 4th day incubated chick embryo. Methodology/Principal Findings: Ischemia was created in chick embryo by ligating the right vitelline artery using sterile surgical suture. Hypoxia inducible factor- 1 alpha (HIF-1a), creatine phospho kinase-MB and reactive oxygen species in animal tissues and cells were measured to confirm ischemia in chick embryo. Additionally, ranolazine, N-acetyl cysteine and trimetazidine were administered as an anti-ischemic drug to validate the present model. Results from the present study depicted that blocking blood flow elevates HIF-1a, lipid peroxidation, peroxynitrite level in ischemic vessels while ranolazine administration partially attenuates ischemia driven HIF-1a expression. Endothelial cell incubated on ischemic blood vessels elucidated a higher level of HIF-1a expression with time while ranolazine treatment reduced HIF-1a in ischemic cells. Incubation of caprine heart strip on chick embryo ischemia model depicted an elevated creatine phospho kinase-MB activity under ischemic condition while histology of the treated heart sections evoked edema and disruption of myofibril structures. Conclusions/Significance: The present study concluded that chick embryo partial ischemia model can be used as a novel ex vivo model of ischemia. Therefore, the present model can be used parallel with the known in vivo ischemia models in understanding the mechanistic insight of ischemia development and in evaluating the activity of anti-ischemic drug.status: publishe

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (A beta) peptides by decreasing the cleavage of amyloid precursor protein (APP) by beta-secretase, without notably affecting alpha- and gamma-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates