A Reversible Gene-Targeting Strategy Identifies Synthetic Lethal Interactions between MK2 and p53 in the DNA Damage Response In Vivo

A fundamental limitation in devising new therapeutic strategies for killing cancer cells with DNA damaging agents is the need to identify synthetic lethal interactions between tumor-specific mutations and components of the DNA damage response (DDR) in vivo. The stress-activated p38 mitogen-activated protein kinase (MAPK)/MAPKAP kinase-2 (MK2) pathway is a critical component of the DDR network in p53-deficient tumor cells in vitro. To explore the relevance of this pathway for cancer therapy in vivo, we developed a specific gene targeting strategy in which Cre-mediated recombination simultaneously creates isogenic MK2-proficient and MK2-deficient tumors within a single animal. This allows direct identification of MK2 synthetic lethality with mutations that promote tumor development or control response to genotoxic treatment. In an autochthonous model of non-small-cell lung cancer (NSCLC), we demonstrate that MK2 is responsible for resistance of p53-deficient tumors to cisplatin, indicating synthetic lethality between p53 and MK2 can successfully be exploited for enhanced sensitization of tumors to DNA-damaging chemotherapeutics in vivo.National Institutes of Health (U.S.) (Grant ES015339)National Institutes of Health (U.S.) (Grant GM60594)National Institutes of Health (U.S.) (Grant GM59281)National Institutes of Health (U.S.) (Grant CA112967)Janssen Pharmaceutical Ltd.Massachusetts Institute of Technology. Center for Environmental Health Sciences (Core Grant P30-CA14051)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Core Grant ES-002109

Oxytocin and vasopressin levels are decreased in the plasma of male schizophrenia patients

Objective Impaired social functioning and autistic symptoms are characteristics of schizophrenia. The social hormones oxytocin (OT) and arginine-vasopressin (AVP) both modulate social interaction and therefore may be involved in the pathogenesis of schizophrenia. We investigated whether men with schizophrenia show altered OT and AVP levels compared with healthy controls (HC) and whether autism symptoms are associated with OT levels. Methods Forty-one men with non-acute schizophrenia and 45 matched HC were enroled. Schizophrenia was assessed with the Positive and Negative Syndrome Scale (PANSS). Blood samples were collected on 2 days, and plasma OT and AVP levels were measured by ELISA immunoassay. Results The schizophrenia patients had significantly lower plasma OT levels than the HC; a similar trend was found for AVP. Plasma OT levels were associated with severe life events, fewer important attached persons, and a higher score on the PANSS negative scale; the most dominant PANSS items were ‘preoccupation’, ‘emotional withdrawal’, and ‘passive/apathetic social withdrawal’. Conclusion These findings support an association between the social hormones OT and AVP and schizophrenia. We suggest that OT metabolism may be altered in schizophrenia, but other possible causes for decreased plasma OT levels in schizophrenia patients include decreased OT synthesis, mRNA expression, and translation. Especially the ‘autistic’ symptoms of schizophrenia seem to be closely linked to an altered metabolism of OT, the ‘attachment’ hormone

An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model

Background: Down syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic cause of mental retardation in humans. Among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early Alzheimer-like neurodegeneration. Mouse models for DS show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. Early postnatal development of both human and mouse-model DS shows the reduced capability of neuronal precursor cells to generate neurons. The exact molecular cause of this reduction, and the role played by increased dosage of individual HSA21 genes, remain unknown.Results: We have subcutaneously injected mouse pluripotent ES cells containing a single freely segregating supernumerary human chromosome 21 (HSA21) into syngeneic mice, to generate transchromosomic teratomas. Transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. Tumours were grown for 30 days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. When paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mRNA levels for neuron specific (Tubb3) and glia specific (Gfap) genes, relative to euploid controls. Two thirds of transchromosomic tumours also showed a lack of PCR amplification with multiple primers specific for HSA21, which were present in the ES cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of HSA21 genes.Conclusion: We demonstrate that a supernumerary chromosome 21 causes Inhibition of Neuroectodermal Differentiation (INDI) of pluripotent ES cells. The data suggest that trisomy of less than a third of HSA21 genes, in two chromosomal regions, might be sufficient to cause this effect

Bid maintains mitochondrial cristae structure and function and protects against cardiac disease in an integrative genomics study.

Bcl-2 family proteins reorganize mitochondrial membranes during apoptosis, to form pores and rearrange cristae. In vitro and in vivo analysis integrated with human genetics reveals a novel homeostatic mitochondrial function for Bcl-2 family protein Bid. Loss of full-length Bid results in apoptosis-independent, irregular cristae with decreased respiration. Bid-/- mice display stress-induced myocardial dysfunction and damage. A gene-based approach applied to a biobank, validated in two independent GWAS studies, reveals that decreased genetically determined BID expression associates with myocardial infarction (MI) susceptibility. Patients in the bottom 5% of the expression distribution exhibit >4 fold increased MI risk. Carrier status with nonsynonymous variation in Bids membrane binding domain, BidM148T, associates with MI predisposition. Furthermore, Bid but not BidM148T associates with Mcl-1Matrix, previously implicated in cristae stability; decreased MCL-1 expression associates with MI. Our results identify a role for Bid in homeostatic mitochondrial cristae reorganization, that we link to human cardiac disease

Insensitivity of chloroplast gene expression to DNA methylation

Presence and possible functions of DNA methylation in plastid genomes of higher plants have been highly controversial. While a number of studies presented evidence for the occurrence of both cytosine and adenine methylation in plastid genomes and proposed a role of cytosine methylation in the transcriptional regulation of plastid genes, several recent studies suggested that at least cytosine methylation may be absent from higher plant plastid genomes. To test if either adenine or cytosine methylation can play a regulatory role in plastid gene expression, we have introduced cyanobacterial genes for adenine and cytosine DNA methyltransferases (methylases) into the tobacco plastid genome by chloroplast transformation. Using DNA cleavage with methylation-sensitive and methylation-dependent restriction endonucleases, we show that the plastid genomes in the transplastomic plants are efficiently methylated. All transplastomic lines are phenotypically indistinguishable from wild-type plants and, moreover, show no alterations in plastid gene expression. Our data indicate that the expression of plastid genes is not sensitive to DNA methylation and, hence, suggest that DNA methylation is unlikely to be involved in the transcriptional regulation of plastid gene expression

Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition

Laser-Induced Electron Diffraction in Chiral Molecules

Strong laser pulses enable probing molecules with their own electrons. The oscillating electric field tears electrons off a molecule, accelerates them, and drives them back toward their parent ion within a few femtoseconds. The electrons are then diffracted by the molecular potential, encoding its structure and dynamics with angstrom and attosecond resolutions. Using elliptically polarized laser pulses, we show that laser-induced electron diffraction is sensitive to the chirality of the target. The field selectively ionizes molecules of a given orientation and drives the electrons along different sets of trajectories, leading them to recollide from different directions. Depending on the handedness of the molecule, the electrons are preferentially diffracted forward or backward along the light propagation axis. This asymmetry, reaching several percent, can be reversed for electrons recolliding from two ends of the molecule. The chiral sensitivity of laser-induced electron diffraction opens a new path to resolve ultrafast chiral dynamics

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all