The Role of TSC2 and Deptor in Fetal Cortical Development

Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder that results form mutations in the TSC1 or TSC2 genes. TSC is a multisystem hamartoma syndrome with manifestations in the brain, heart, lungs, kidney, skin and eyes. Neurologically, TSC patients may exhibit severe epilepsy, cognitive disabilities, and autism spectrum disorders. TSC1 and TSC2 proteins form a heterodimeric complex that serves to inhibit mammalian target of rapamycin (mTOR) signaling pathway. TSC1 and TSC2 receive activating or inhibitory signaling from multiple inputs including growth factors, insulin signaling, energy and amino acid levels, and proinflammatory pathways, and then integrate those signals to regulate the activity of mTOR. mTOR signaling plays a critical role in regulating cell growth, transcription, translation, and autophagy. Animal models have shed light on certain features of TSC, but failed to recapitulate the disease completely and currently further research is under way to better understand this devastating disorder. To date, mTOR signaling hyperactivation has been demonstrated in TSC tubers at postnatal time points, thus we set out to study the profile of mTOR activation in the fetal brain. We utilized both mouse neural progenitors in vitro and developing brain in vivo systems to understand the effects of Tsc1 and Tsc2 during brain development. Furthermore, after the identification of a new mTOR regulatory protein Deptor (DEPDC6 gene), which inhibits the mTORC1 and mTORC2 signaling pathways similar to TSC1-TSC protein complex, we examined its role in brain development. We found that Deptor shRNA knockdown results in mTORC1 and mTORC2 activation in vitro as well as abnormal migration in vivo. Our results show that mTOR signaling pathway could be the common pathway on which TSC1, TSC2, and DEPTOR converge and exert their effects on brain development. These results suggest mTOR signaling and its downstream effectors could be targets for therapeutic treatment during embryogenesis and could potentially prevent abnormal brain development

PERFORMANCE OF SOCIALLY RESPONSIBLE INDICES DURING MARKET CRISIS IN NORTH AMERICA AND EUROPE

This paper investigates whether socially responsible investment indices in the United States, Canada, the Eurozone and the United Kingdom provide downside protection during market crisis when compared to their respective market indices. Socially responsible investment indices in US and Canada perform similarly to their market indices during market crisis periods between 2000 and 2014, offering neither downside protection nor excess return in overall market conditions.   In Eurozone, the socially responsible investment index we selected performs worse than their market index during both the Financial Crisis and the Euro Crisis but not during the Tech Bubble. In the United Kingdom, socially responsible investment index underperforms its respective market index during all crisis periods, including the Tech Bubble, Financial Crisis and the Euro Crisis but outperforms during non-crisis periods. Overall, we do not find that SRI indices offer downmarket protection in North America and Europe

Acetylation at lysine 86 of escherichia coli HUβ modulates the DNA-binding capability of the protein

DNA-binding protein HU is highly conserved in bacteria and has been implicated in a range of cellular processes and phenotypes. Like eukaryotic histones, HU is subjected to post-translational modifications. Specifically, acetylation of several lysine residues have been reported in both homologs of Escherichia coli HU. Here, we investigated the effect of acetylation at Lys67 and Lys86, located in the DNA binding-loop and interface of E. coli HUβ, respectively. Using the technique of genetic code expansion, homogeneous HUβ(K67ac) and HUβ(K86ac) protein units were obtained. Acetylation at Lys86 seemed to have negligible effects on protein secondary structure and thermal stability. Nevertheless, we found that this site-specific acetylation can regulate DNA binding by the HU homodimer but not the heterodimer. Intriguingly, while Lys86 acetylation reduced the interaction of the HU homodimer with short double-stranded DNA containing a 2-nucleotide gap or nick, it enhanced the interaction with longer DNA fragments and had minimal effect on a short, fully complementary DNA fragment. These results demonstrate the complexity of post-translational modifications in functional regulation, as well as indicating the role of lysine acetylation in tuning bacterial gene transcription and epigenetic regulation

Effect of membrane fusion protein AdeT1 on the antimicrobial resistance of Escherichia coli

Acinetobacter baumannii is a prevalent pathogen that can rapidly acquire resistance to antibiotics. Indeed, multidrug-resistant A. baumannii is a major cause of hospital-acquired infections and has been recognised by the World Health Organization as one of the most threatening bacteria to our society. Resistance-nodulation-division (RND) type multidrug efflux pumps have been demonstrated to convey antibiotic resistance to a wide range of pathogens and are the primary resistance mechanism employed by A. baumannii. A component of an RND pump in A. baumannii, AdeT1, was previously demonstrated to enhance the antimicrobial resistance of Escherichia coli. Here, we report the results of experiments which demonstrate that wild-type AdeT1 does not confer antimicrobial resistance in E. coli, highlighting the importance of verifying protein production when determining minimum inhibitory concentrations (MICs) especially by broth dilution. Nevertheless, using an agar-based MIC assay, we found that propionylation of Lys280 on AdeT1 renders E. coli cells more resistant to erythromycin

Development of mammalian cell logic gates controlled by unnatural amino acids

Genetic code expansion employing an orthogonal aminoacyl-tRNA synthetase/tRNA pair for site-specific unnatural amino acid incorporation has a wide range of applications. However, a blank (orthogonal) codon is needed for each orthogonal tRNA. Although the amber stop codon is commonly used as the blank codon, the use of stop codons as the blank codons inevitably limits the number of possible different unnatural amino acids to be incorporated into proteins in a single cell. To overcome this limitation, we evaluated the capability of 11 Pyl tRNA variants for decoding quadruplet codons in mammalian cells. With efficient quadruplet-decoding orthogonal tRNA variants available, it is possible for simultaneous incorporation of more than three different unnatural amino acids in mammalian cells

Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus

Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, \u3e10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

Progress in targeted gene editing by programmable endonucleases has paved the way for their use in gene therapy. Particularly, Cas9 is an endonuclease with high activity and flexibility, rendering it an attractive option for therapeutic applications in clinical settings. Many disease-causing mutations could potentially be corrected by this versatile new technology. In addition, recently developed switchable Cas9 variants, whose activity can be controlled by an external stimulus, provide an extra level of spatiotemporal control on gene editing and are particularly desirable for certain applications. Here, we discuss the considerations and difficulties for implementing Cas9 to in vivo gene therapy. We put particular emphasis on how switchable Cas9 variants may resolve some of these barriers and advance gene therapy in the clinical setting

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

Progress in targeted gene editing by programmable endonucleases has paved the way for their use in gene therapy. Particularly, Cas9 is an endonuclease with high activity and flexibility, rendering it an attractive option for therapeutic applications in clinical settings. Many disease-causing mutations could potentially be corrected by this versatile new technology. In addition, recently developed switchable Cas9 variants, whose activity can be controlled by an external stimulus, provide an extra level of spatiotemporal control on gene editing and are particularly desirable for certain applications. Here, we discuss the considerations and difficulties for implementing Cas9 to in vivo gene therapy. We put particular emphasis on how switchable Cas9 variants may resolve some of these barriers and advance gene therapy in the clinical setting

Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome).

We report an allelic series of eight mutations in GATA2 underlying Emberger syndrome, an autosomal dominant primary lymphedema associated with a predisposition to acute myeloid leukemia. GATA2 is a transcription factor that plays an essential role in gene regulation during vascular development and hematopoietic differentiation. Our findings indicate that haploinsufficiency of GATA2 underlies primary lymphedema and predisposes to acute myeloid leukemia in this syndrome