access to regiodifferentiated meta-amino
phenols is described. The strategy relies upon distinct deprotonation
conditions to afford regioisomeric thermodynamic and kinetic dienes
that undergo a tandem Diels–Alder and retro-Diels–Alder sequence with assorted acetylenic dienophiles
to afford a range of aromatic products.
In plants, microRNAs (miRNAs) play essential roles in growth, development, yield, stress response and interactions with pathogens. However no miRNA has been experimentally documented to be functionally involved in fruit ripening although many miRNAs have been profiled in fruits. Here we show that SlymiR157 and SlymiR156 differentially modulate ripening and softening in tomato (Solanum lycopersicum). SlymiR157 is expressed and developmentally regulated in normal tomato fruits and in those of the Colourless non-ripening (Cnr) epimutant. It regulates expression of the key ripening gene LeSPL-CNR in a likely dose-dependent manner through miRNA-induced mRNA degradation and translation repression. Viral delivery of either pre-SlymiR157 or mature SlymiR157 results in delayed ripening. Furthermore, qRT-PCR profiling of key ripening regulatory genes indicates that the SlymiR157-target LeSPL-CNR may affect expression of LeMADS-RIN, LeHB1, SlAP2a and SlTAGL1. However SlymiR156 does not affect the onset of ripening, but it impacts fruit softening after the red ripe stage. Our findings reveal that working together with a ripening network of transcription factors, SlymiR157 and SlymiR156 form a critical additional layer of regulatory control over the fruit ripening process in tomato.
In contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.
Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3′ ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP−/− mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.
Downregulation and mutations of the nuclear-architecture proteins Lamin A and C cause misshapen nuclei and altered chromatin organization associated with cancer and laminopathies, including the premature-aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we identified the small molecule “Remodelin” that improved nuclear architecture, chromatin organization and fitness of both human Lamin A/C depleted cells and HGPS-derived patient cells, and decreased markers of DNA damage in these cells. Using a combination of chemical, cellular and genetic approaches, we identified the acetyl-transferase protein NAT10 as the target of Remodelin that mediated nuclear shape rescue in laminopathic cells via microtubule reorganization. These findings provide insights into how NAT10 affects nuclear architecture, and suggest alternative strategies for treating laminopathies and aging.
Nucleic acid-functionalized gold surfaces have been used extensively for the development of biological sensors. The development of an effective biomarker detection assay requires careful design, synthesis and characterization of probe components. In this feature article, we describe fundamental probe development constraints and provide a critical appraisal of the current methodologies and applications in the field. We discuss critical issues and obstacles that impede the sensitivity and reliability of the sensors to underscore the challenges that must be met to advance the field of biomarker detection.
Gold nanoparticles; nucleic acid sensors; hAuNPs; electrochemistry; biobarcode assay; aptamers; DNAzymes
Tourette syndrome (TS) is a developmental neurological disorder characterized by vocal and motor tics  and associated with cortical-striatal-thalamic-cortical circuit dysfunction [2, 3], hyperexcitability within cortical motor areas , and altered intracortical inhibition [4–7]. TS often follows a developmental time course in which tics become increasingly more controlled during adolescence in many individuals , who exhibit enhanced control over their volitional movements [8–11]. Importantly, control over motor outputs appears to be brought about by a reduction in the gain of motor excitability [6, 7, 12, 13]. Here we present a neurochemical basis for a localized gain control mechanism. We used ultra-high-field (7 T) magnetic resonance spectroscopy to investigate in vivo concentrations of γ-aminobutyric acid (GABA) within primary and secondary motor areas of individuals with TS. We demonstrate that GABA concentrations within the supplementary motor area (SMA)—a region strongly associated with the genesis of motor tics in TS —are paradoxically elevated in individuals with TS and inversely related to fMRI blood oxygen level-dependent activation. By contrast, GABA concentrations in control sites do not differ from those of a matched control group. Importantly, we also show that GABA concentrations within the SMA are inversely correlated with cortical excitability in primary motor cortex and are predicted by motor tic severity and white-matter microstructure (FA) within a region of the corpus callosum that projects to the SMA within each hemisphere. Based upon these findings, we propose that extrasynaptic GABA contributes to a form of control, based upon localized tonic inhibition within the SMA, that may lead to the suppression of tics.
•We report a 7 T 1H MRS investigation of GABA in Tourette syndrome (TS)•GABA levels within the SMA are significantly elevated in TS•SMA GABA is negatively correlated with SMA BOLD and cortical excitability•SMA GABA is predicted by motor tic severity and corpus callosum FA values
Using magnetic resonance spectroscopy, Draper et al. find that concentrations of the neurotransmitter GABA in motor areas of the brain are associated with symptom severity in individuals with Tourette syndrome.
Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that alters cortical excitability in a polarity specific manner and has been shown to influence learning and memory. tDCS may have both on-line and after-effects on learning and memory, and the latter are thought to be based upon tDCS-induced alterations in neurochemistry and synaptic function. We used ultra-high-field (7 T) magnetic resonance spectroscopy (MRS), together with a robotic force adaptation and de-adaptation task, to investigate whether tDCS-induced alterations in GABA and Glutamate within motor cortex predict motor learning and memory. Note that adaptation to a robot-induced force field has long been considered to be a form of model-based learning that is closely associated with the computation and ‘supervised’ learning of internal ‘forward’ models within the cerebellum. Importantly, previous studies have shown that on-line tDCS to the cerebellum, but not to motor cortex, enhances model-based motor learning. Here we demonstrate that anodal tDCS delivered to the hand area of the left primary motor cortex induces a significant reduction in GABA concentration. This effect was specific to GABA, localised to the left motor cortex, and was polarity specific insofar as it was not observed following either cathodal or sham stimulation. Importantly, we show that the magnitude of tDCS-induced alterations in GABA concentration within motor cortex predicts individual differences in both motor learning and motor memory on the robotic force adaptation and de-adaptation task.
•Ultra-high-field (7 T) magnetic resonance spectroscopy study of the effects of tDCS.•Anodal tDCS leads to a polarity and site specific reduction in MRS-GABA.•tDCS-induced changes in MRS-GABA in M1 predict model-based motor learning/memory.
BOLD, blood-oxygen-level-dependent; fMRI, functional magnetic resonance imaging; GABA, γ-amino-butyric acid; M1, primary motor cortex; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; NAA, N-acetylaspartate; NAAG, N-acetylaspartylglutamate; tDCS, transcranial direct current stimulation; TMS, transcranial magnetic stimulation; V1, primary visual cortex; Motor learning; Force adaptation; Magnetic resonance spectroscopy; tDCS; GABA
We use micropatterning
and strain engineering to encapsulate single
living mammalian cells into transparent tubular architectures consisting
of three-dimensional (3D) rolled-up nanomembranes. By using optical
microscopy, we demonstrate that these structures are suitable for
the scrutiny of cellular dynamics within confined 3D-microenvironments.
We show that spatial confinement of mitotic mammalian cells inside
tubular architectures can perturb metaphase plate formation, delay
mitotic progression, and cause chromosomal instability in both a transformed
and nontransformed human cell line. These findings could provide important
clues into how spatial constraints dictate cellular behavior and function.
Nanomembranes; rolled-up nanotechnology; mitosis; 3D cell culture
scaffold; spatial confinement; chromosome segregation
The DNA damage response (DDR) is critical for genome stability and the suppression of a wide variety of human malignancies, including neurodevelopmental disorders, immunodeficiency, and cancer. In addition, the efficacy of many chemotherapeutic strategies is dictated by the status of the DDR. Ubiquitin-specific protease 28 (USP28) was reported to govern the stability of multiple factors that are critical for diverse aspects of the DDR. Here, we examined the effects of USP28 depletion on the DDR in cells and in vivo. We found that USP28 is recruited to double-strand breaks in a manner that requires the tandem BRCT domains of the DDR protein 53BP1. However, we observed only minor DDR defects in USP28-depleted cells, and mice lacking USP28 showed normal longevity, immunological development, and radiation responses. Our results thus indicate that USP28 is not a critical factor in double-strand break metabolism and is unlikely to be an attractive target for therapeutic intervention aimed at chemotherapy sensitization.
The microbiota of four individual deep water sponges, Lissodendoryx diversichela, Poecillastra compressa, Inflatella pellicula, and Stelletta normani, together with surrounding seawater were analysed by pyrosequencing of a region of the 16S rRNA gene common to Bacteria and Archaea. Due to sampling constraints at depths below 700 m duplicate samples were not collected. The microbial communities of L. diversichela, P. compressa and I. pellicula were typical of low microbial abundance (LMA) sponges while S. normani had a community more typical of high microbial abundance (HMA) sponges. Analysis of the deep sea sponge microbiota revealed that the three LMA-like sponges shared a set of abundant OTUs that were distinct from those associated with sponges from shallow waters. Comparison of the pyrosequencing data with that from shallow water sponges revealed that the microbial communities of all sponges analysed have similar archaeal populations but that the bacterial populations of the deep sea sponges were distinct. Further analysis of the common and abundant OTUs from the three LMA-like sponges placed them within the groups of ammonia oxidising Archaea (Thaumarchaeota) and sulphur oxidising γ-Proteobacteria (Chromatiales). Reads from these two groups made up over 70% of all 16S rRNA genes detected from the three LMA-like sponge samples, providing evidence of a putative common microbial assemblage associated with deep sea LMA sponges.
A combination of RNase- and detergent-based preextraction with high-resolution microscopy allows the detection of Ku and other DNA repair proteins at single double-strand breaks in cells.
DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents.
Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.
The mouse germ-line presents a unique opportunity to study epigenetic reprogramming in vivo1. Recently we showed that genome-wide active DNA demethylation in primordial germ cells (PGCs) is linked to changes in nuclear architecture and extensive loss of histone modifications brought about by widespread histone replacement2. Notably, this chromatin remodelling follows the onset of genome-wide DNA demethylation, which raises a possibility that DNA demethylation may be linked to a DNA repair process2. Here we show the activation of components of the base excision repair (BER) pathway and the presence of single-strand DNA (ssDNA) breaks at the time of genome-wide DNA demethylation in PGCs. We found high levels of expression of critical BER components as well as chromatin-bound XRCC1 together with nuclear poly-ADP-ribosylation (PAR), specifically at the time when PGCs are undergoing DNA demethylation. A similar wave of genome-wide DNA demethylation occurs in the zygote affecting only the paternal genome3-5 where we observed a strikingly similar activation of BER components. Notably, maternally inherited Stella promotes this epigenetic asymmetry, since in zygotes lacking this protein, DNA demethylation is detected in both pronuclei. Crucially, zygotes lacking Stella exhibit aberrant targeting of active BER to both pronuclei. Finally we demonstrate that small molecule inhibitors of diverse BER components administered during in vitro fertilisation interfere with the progress of DNA demethylation.
Our combined observations demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation and provides a vital mechanistic link to the extensive chromatin remodelling in developing PGCs2.
Detecting genomic changes represents a critical step in cellular responses to DNA damage. Here, we show that tyrosine phosphorylation of the protein acetyltransferase KAT5 (Tip60) increases in response to DNA damage in a manner that promotes KAT5 binding to the histone mark H3K9me3. This in turn triggers KAT5-mediated acetylation of the ATM kinase, promoting DNA-damage checkpoint activation and cell survival. We also establish that chromatin alterations per se can enhance KAT5 tyrosine phosphorylation and ATM-dependent signaling, and identify the proto-oncogene c-Abl as mediating this modification. These findings define KAT5 as a key sensor for genomic and chromatin perturbations, and highlight a prime role for c-Abl in such events.
Genotypic strains of mutans streptococci (MS) may vary in important virulence properties, and may be differentially affected by specific components of full-mouth caries restorative therapy. The purpose of this pilot study was to identify MS strains that predominate following caries restorative therapy.
Plaque from seven children with severe early childhood caries was collected before and following therapy. MS isolates (N=828) were subjected to polymerase chain reaction (PCR), and arbitrarily primed-PCR (AP-PCR) for assignment within MS strains. Determining the longitudinal changes in MS strain distribution over time within each patient required the isolation of larger numbers of isolates per patient, but from fewer patients.
Up to 39 genotypic strains of S. mutans and S. sobrinus, and seven genotypic strains of non-MS streptococci were identified by AP-PCR and 16S ribosomal rRNA gene sequencing. The number of MS strains isolated from each patient were 3–7 prior to treatment, diminishing to 1–2 dominant MS strains in most patients 6 months post-therapy.
Caries restorative therapy resulted in shifts of specific MS and non-MS streptococci strains. The implications are that caries restorative therapy affects the distribution of MS strains, and that well-accepted practices for caries prevention should be more closely examined for efficacy.
mutans streptococci; selection and distribution of genotypic MS strains; Streptococcus mutans; oral streptococci; severe early childhood caries; caries restorative therapy
Patients with alien hand syndrome (AHS) experience making apparently deliberate and purposeful movements with their hand against their will. However, the mechanisms contributing to these involuntary actions remain poorly understood. Here, we describe two experimental investigations in a patient with corticobasal syndrome (CBS) with alien hand behaviour in her right hand. First, we show that responses with the alien hand are made significantly more quickly to images of objects which afford an action with that hand compared to objects which afford an action with the unaffected hand. This finding suggests that involuntary grasping behaviours in AHS might be due to exaggerated, automatic motor activation evoked by objects which afford actions with that limb. Second, using a backwards masked priming task, we found normal automatic inhibition of primed responses in the patient's unaffected hand, but importantly there was no evidence of such suppression in the alien limb. Taken together, these findings suggest that grasping behaviours in AHS may result from exaggerated object affordance effects, which might potentially arise from disrupted inhibition of automatically evoked responses.
Alien limb; Object affordance; Automatic inhibition; Masked priming
Cognitive performance is known to change over age 45, especially processing speed. Studies to date indicate that change in performance with ageing is largely environmentally mediated, with little contribution from genetics. We estimated the heritability of a longitudinal battery of computerised cognitive tests including speed measures, using a classical twin design. 324 (127 MZ, 197 DZ) female twins, aged 43–73 at baseline testing, were followed-up after 10 years, using seven measures of the Cambridge Automated Neuropsychological Test battery, four of which were measures of response latency (speed). Results were analysed using univariate and bivariate structural equation modelling. Heritability of longitudinal change was found in 5 of the 7 tests, ranging from 21 to 41 %. The genetic aetiology was remarkably stable. The first principle component of change was strongly associated with age (p < 0.001) and heritable at 47 % (27–62 %). While estimates for heritability increased in all measures over time compared to baseline, these increases were statistically non-significant. This computerised battery showed significant heritability of age-related change in cognition. Focus on this form of change may aid the search for genetic pathways involved in normal and pre-morbid cognitive ageing.
Electronic supplementary material
The online version of this article (doi:10.1007/s10519-013-9612-z) contains supplementary material, which is available to authorized users.
Processing speed; Heritability; Cognition; Aging; Twin study; Accelerating change
The histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells.
Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium.
folate receptor alpha; FRA; ovarian cancer; fallopian tube; 1-carbon metabolism; reduced folate carrier; methylation
Covalent post-translational modification of proteins by ubiquitin and ubiquitin-like factors has emerged as a general mechanism to regulate myriad intra-cellular processes. The addition and removal of ubiquitin or ubiquitin-like proteins from factors has recently been demonstrated as a key mechanism to modulate DNA damage response (DDR) pathways. It is thus, timely to evaluate the potential for ubiquitin pathway enzymes as DDR drug targets for therapeutic intervention. The synthetic lethal approach provides exciting opportunities for the development of targeted therapies to treat cancer: most tumours have lost critical DDR pathways, and thus rely more heavily on the remaining pathways, while normal tissues are still equipped with all DDR pathways. Here, we review key deubiquitylating enzymes (DUBs) involved in DDR pathways, and describe how targeting DUBs may lead to selective therapies to treat cancer patients.
Synthetic lethality; Ubiquitin; Deubiquitylating enzyme; DUB; DNA damage response; DNA repair; Drug discovery; Checkpoint control
cytokinesis; Dma1; Dma2; E3 ubiquitin-ligase; histone modification; RNF8; Septins; Septin-filament; ubiquitin
Previous work has established that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is stabilized in an ATM-dependent manner in response to DNA damage and acts as a cofactor for p53-mediated transcription. Here, we show that in response to DNA damage caused by ionizing radiation, hnRNP K is phosphorylated in an ATM-dependent manner. Furthermore, our data indicate that ATM-dependent hnRNP K phosphorylation is required for its stabilization and its function as a p53 transcriptional cofactor in response to DNA damage. These findings thereby establish hnRNP K as an ATM target and help define how ATM orchestrates p53-dependent transcriptional responses in response to genotoxic stress.
DNA damage; ATM; phosphorylation; transcription; p53
The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA damaging agents, thus suggesting an important link between RNA metabolism and DNA repair. Our results broaden the knowledge of DNA damage signaling networks and identify novel components of the DDR.
The conserved MRE11-RAD50-NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target for phosphorylation by cyclin-dependent kinases (CDKs). We show that NBS1 serine 432 (Ser-432) phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification of NBS1 stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of this phosphorylation not only negatively affects DSB repair by HR but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.
NBS1; cyclin-dependent kinase; homologous recombination; end joining; replication restart