Autism spectrum disorders (ASD) are hypothesized to originate in utero from perturbations in neural stem cell niche regions of the developing brain. Dynamic epigenetic processes including DNA methylation are integral to coordinating typical brain development. However, the extent and consequences of alterations to DNA methylation states in neural stem cell compartments in ASD are unknown. Here, we report significant DNA methylation defects in the subventricular zone of the lateral ventricles from postmortem brain of 17 autism diagnosed compared to 17 age- and gender-matched typically developing individuals. Both array- and sequencing-based genome-wide methylome analyses independently revealed that these alterations were preferentially targeted to intragenic and bivalently modified chromatin domains of genes predominately involved in neurodevelopment, which associated with aberrant precursor messenger RNA splicing events of ASD-relevant genes. Integrative analysis of our ASD and typically developing postmortem brain methylome datasets with that from fetal brain at different neurodevelopmental stages revealed that the methylation states of differentially methylated loci associated with ASD remarkably resemble the methylation states at earlier time points in fetal brain development. This observation was confirmed using additional methylome datasets from three other brain regions. Altogether, these findings implicate an epigenetic delay in the trajectory of normal DNA methylation states during the course of brain development that may consequently lead to deleterious transcriptomic events in ASD and support the hypothesis of an early developmental origin of ASD.
Cloning and expression plasmids are the workhorses of modern molecular biology. Despite the pathway paved by synthetic biology, laboratories around the globe still relay on standard cloning techniques using plasmids with reporter proteins for positive clone selection, such as β-galactosidase alpha peptide complementation for blue/white screening or ccdB, which encodes for a toxic DNA gyrase. These reporters, when interrupted, serve as a positive clone detection system. In the present report, we show that molecular cloning plasmids bearing the coding sequence for a 25.4 kDa protein, AmilCP, encoded by a 685 bp gene, that is well expressed in Escherichia coli, render blue-purple colonies. Using this reporter protein, we developed and tested a cloning system based on the constitutive expression of the non-toxic AmilCP protein, that once interrupted, the loss of purple color serves to facilitate positive clone selection. The main advantage of this system is that is less expensive than other systems since media do not contain chromogenic markers such as X-gal, which is both expensive and cumbersome to prepare and use, or inductors such as IPTG. We also designed an inducible expression plasmid suitable for recombinant protein expression that also contains AmilCP cloning selection marker, a feature not commonly found in protein expression plasmids. The use of chromogenic reporters opens an important avenue for its application in other organisms besides E. coli for clone selection or even for mutant selection.
Aim: As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial compounds is necessary. One strategy is to generate treatment alternatives by drug repurposing. Methods and Results: In this work, phenotypic microarrays were used for the screening of miscellaneous compounds against the growth and biofilm formation of Acinetobacter baumannii, an important emergent multidrugresistant opportunistic pathogen. The results showed that the phenothiazine derivatives, such as promethazine, trifluoperazine, thioridazine, and chlorpromazine, inhibited the growth of antibiotic-sensitive and multidrugresistant strains (showing minimal inhibitory concentrations ranging from 0Á05 to 0Á6 g l À1 and minimal bactericidal concentrations ranging from 0Á1 to 2Á5 g l À1 ). All phenothiazine derivatives were active against biofilm cells (with minimal biofilm eradication concentrations ranging from 0Á5 to >3 g l À1 ). Chlorpromazine promoted reactive oxigen species (ROS) production, and cell membrane and DNA damage. Chlorpromazine showed synergy with antibiotics such as ceftazidime, meropenem, and colistin and was an effective treatment for experimentally infected Galleria mellonella when combined with ceftazidime. Conclusions: It was demonstrated that phenothiazine derivatives, especially chlorpromazine, are drugs with attractive antibacterial properties against nosocomial MDR strains of A. baumannii, by generating ROS and cell membrane and DNA damage. Significance and Impact of the Study: The present study indicates that repurposing phenothiazine derivatives for treating recalcitrant infections by A. baumannii could be promising.
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