Question: MicroRNA participates in the control of Neural Stem Cell (NSC) differentiation and proliferation balance.

Epigenetic Control of miR-184 by MBD1 Influences Neural Stem Cell Proliferation and Differentiation Dynamics.
Methyl-CpG binding protein 1 (MBD1) plays a critical role in modulating gene expression through DNA methylation-based epigenetic mechanisms. Our previous studies revealed that a deficiency in MBD1 disrupts the differentiation and neurogenesis of adult neural stem/progenitor cells (aNSCs), but the exact molecular pathway was not fully understood. In this work, we uncover that MBD1 exerts control over the expression of several microRNAs in aNSCs, notably highlighting the suppression of miR-184 by MBD1. Elevated miR-184 expression was found to enhance aNSC proliferation while simultaneously suppressing their differentiation, whereas the downregulation of miR-184 alleviated the adverse effects associated with MBD1 insufficiency. We also identified that miR-184 governs the expression of Numblike (Numbl), a key factor in brain development, by interacting with the 3'-UTR of Numbl mRNA and influencing its translational process. The overexpression of exogenous Numbl was able to counteract the defects in aNSCs caused by either excessive miR-184 levels or MBD1 insufficiency. Consequently, the interplay between MBD1, miR-184, and Numbl constitutes a regulatory network that is pivotal in maintaining the equilibrium between aNSC proliferation and differentiation.

Glioblastoma multiforme (GBM) is a fatal malignancy of the central nervous system, commonly associated with chemoresistance. The alkylating agent Temozolomide (TMZ) is the front-line chemotherapeutic agent and has undergone intense studies on resistance. These studies reported on mismatch repair gene upregulation, ABC-targeted drug efflux, and cell cycle alterations. The mechanism by which TMZ induces cell cycle arrest has not been well-established. TMZ-resistant GBM cells have been linked to microRNA (miRNA) and exosomes. A cell cycle miRNA array identified distinct miRNAs only in exosomes from TMZ-resistant GBM cell lines and primary spheres. We narrowed the miRs to miR-93 and -193 and showed in computational analyses that they could target Cyclin D1. Since Cyclin D1 is a major regulator of cell cycle progression, we performed cause-effect studies and showed a blunting effects of miR-93 and -193 in Cyclin D1 expression. These two miRs also decreased cell cycling quiescence and induced resistance to TMZ. Taken together, our data provide a mechanism by which GBM cells can exhibit TMZ-induced resistance through miRNA targeting of Cyclin D1. The data provide a number of therapeutic approaches to reverse chemoresistance at the miRNA, exosomal and cell cycle points.

Allergic airway inflammation is characterized by marked in situ changes in gene and protein expression, yet the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in this process has not yet been reported. Using a highly sensitive microarray-based approach, we identified 21 miRNAs with differential expression between doxycycline-induced lung-specific IL-13 transgenic mice (with allergic airway inflammation) and control mice. In particular, we observed overexpression of miR-21 and underexpression of miR-1 in the induced IL-13 transgenic mice compared with control mice. These findings were validated in two independent models of allergen-induced allergic airway inflammation and in IL-4 lung transgenic mice. Although IL-13-induced miR-21 expression was IL-13Ralpha1 dependent, allergen-induced miR-21 expression was mediated mainly independent of IL-13Ralpha1 and STAT6. Notably, predictive algorithms identified potential direct miR-21 targets among IL-13-regulated lung transcripts, such as IL-12p35 mRNA, which was decreased in IL-13 transgenic mice. Introduction of pre-miR-21 dose dependently inhibited cellular expression of a reporter vector harboring the 3'-untranslated region of IL-12p35. Moreover, mutating miR-21 binding sites in IL-12p35 3'-untranslated region abrogated miR-21-mediated repression. In summary, we have identified a miRNA signature in allergic airway inflammation, which includes miR-21 that modulates IL-12, a molecule germane to Th cell polarization.

Perturbations in neuregulin-1 (NRG1)/ErbB4 function have been associated with schizophrenia. Affected patients exhibit altered levels of these proteins and display hypofunction of glutamatergic synapses as well as altered neuronal circuitry. However, the role of NRG1/ErbB4 in regulating synapse maturation and neuronal process formation has not been extensively examined. Here we demonstrate that ErbB4 is expressed in inhibitory interneurons at both excitatory and inhibitory postsynaptic sites. Overexpression of ErbB4 postsynaptically enhances size but not number of presynaptic inputs. Conversely, knockdown of ErbB4 using shRNA decreases the size of presynaptic inputs, demonstrating a specific role for endogenous ErbB4 in synapse maturation. Using ErbB4 mutant constructs, we demonstrate that ErbB4-mediated synapse maturation requires its extracellular domain, whereas its tyrosine kinase activity is dispensable for this process. We also demonstrate that depletion of ErbB4 decreases the number of primary neurites and that stimulation of ErbB4 using a soluble form of NRG1 results in exuberant dendritic arborization through activation of the tyrosine kinase domain of ErbB4 and the phosphoinositide 3-kinase pathway. These findings demonstrate that NRG1/ErbB4 signaling differentially regulates synapse maturation and dendritic morphology via two distinct mechanisms involving trans-synaptic signaling and tyrosine kinase activity, respectively.

Zebrafish maintain a greater capacity than mammals for central nervous system repair after injury. Understanding differences in regenerative responses between different vertebrate species may shed light on mechanisms to improve repair in humans. Quinolinic acid is an excitotoxin that has been used to induce brain injury in rodents for modeling Huntington's disease and stroke. When injected into the adult rodent striatum, this toxin stimulates subventricular zone neurogenesis and neuroblast migration to injury. However, most new neurons fail to survive and lesion repair is minimal. We used quinolinic acid to lesion the adult zebrafish telencephalon to study reparative processes. We also used conditional transgenic lineage mapping of adult radial glial stem cells to explore survival and integration of neurons generated after injury. Telencephalic lesioning with quinolinic acid, and to a lesser extent vehicle injection, produced cell death, microglial infiltration, increased cell proliferation, and enhanced neurogenesis in the injured hemisphere. Lesion repair was more complete with quinolinic acid injection than after vehicle injection. Fate mapping of her4-expressing radial glia showed injury-induced expansion of radial glial stem cells that gave rise to neurons which migrated to injury, survived at least 8 weeks and formed long-distance projections that crossed the anterior commissure and synapsed in the contralateral hemisphere. These findings suggest that quinolinic acid lesioning of the zebrafish brain stimulates adult neural stem cells to produce robust regeneration with long-distance integration of new neurons. This model should prove useful for elucidating reparative mechanisms that can be applied to restorative therapies for mammalian brain injury.

MicroRNAs (miRNAs) play crucial roles during the occurrence and development of gastric cancer. Conventional serological tests for screening gastric cancer have limits on sensitivity and specificity. Several miRNAs in peripheral blood have been used as biomarkers of gastric cancer. However, most of these miRNAs are shared by several types of cancer. Thanks to the tissue specificity of gastric juice, here we examined the feasibility of using gastric juice miR-129-1/2, which are aberrantly expressed in gastric cancer, to screen gastric cancer. Total of 141 gastric juices samples from gastric cancer, gastric ulcer, atrophic gastritis, and minimal gastritis patients or subjects with normal mucosa were collected by gastroscopy. The gastric juice miR-129-1/2 levels were detected by quantitative reverse transcription-polymerase chain reaction. A receiver operating characteristic (ROC) curve was constructed for differentiating patients with gastric cancer from patients with benign gastric diseases. We showed that, compared with patients with benign gastric diseases, patients with gastric cancer had significantly lower levels of gastric juice miR-129-1-3p and miR-129-2-3p. The areas under ROC curve (AUC) were 0.639 and 0.651 for miR-129-1-3p and miR-129-2-3p, respectively. Using the parallel combination test, the AUC was up to 0.656. In summary, our results suggest that gastric juice miR-129-1-3p and miR-129-2-3p are potential biomarkers for the screening gastric cancer, and the detection of gastric juice miRNAs is a convenient non-invasion method for the diagnosis of gastric cancer.

Deciphering the molecular basis of stem cell pluripotency is fundamental to the understanding of stem cell biology, early embryonic development, and to the clinical application of regenerative medicine. We report here that the molecular chaperone heat shock protein 90 (Hsp90) is essential for mouse embryonic stem cell (ESC) pluripotency through regulating multiple pluripotency factors, including Oct4, Nanog, and signal transducer and activator of transcription 3. Inhibition of Hsp90 by either 17-N-Allylamino-17-demethoxygeldanamycin or miRNA led to ESC differentiation. Overexpression of Hsp90β partially rescued the phenotype; in particular, the levels of Oct4 and Nanog were restored. Notably, Hsp90 associated with Oct4 and Nanog in the same cellular complex and protected them from degradation by the ubiquitin proteasome pathway, suggesting that Oct4 and Nanog are potential novel Hsp90 client proteins. In addition, Hsp90 inhibition reduced the mRNA level of Oct4, but not that of Nanog, indicating that Hsp90 participates in Oct4 mRNA processing or maturation. Hsp90 inhibition also increased expression of some protein markers for mesodermal lineages, implying that Hsp90 suppresses mesodermal differentiation from ESCs. These findings support a new role for Hsp90 in maintaining ESC pluripotency by sustaining the level of multiple pluripotency factors, particularly Oct4 and Nanog.

Despite the recent identification of the transcriptional regulatory circuitry involving SOX2, NANOG, and OCT-4, the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here, we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency, prevents cell proliferation, and enhances mesoderm and endoderm activities in hESCs. At the molecular level, mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growth-inhibitory genes, as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration.

During development, cells monitor and adjust their rates of accumulation to produce organs of predetermined size. We show here that central nervous system-specific deletion of the essential adherens junction gene, alphaE-catenin, causes abnormal activation of the hedgehog pathway, resulting in shortening of the cell cycle, decreased apoptosis, and cortical hyperplasia. We propose that alphaE-catenin connects cell-density-dependent adherens junctions with the developmental hedgehog pathway and that this connection may provide a negative feedback loop controlling the size of developing cerebral cortex.

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.

Cadherins are Ca(2+)-dependent cell-cell adhesion molecules that play critical roles in animal morphogenesis. Various cadherin-related molecules have also been identified, which show diverse functions, not only for the regulation of cell adhesion but also for that of cell proliferation and planar cell polarity. During the past decade, understanding of the roles of these molecules in the nervous system has significantly progressed. They are important not only for the development of the nervous system but also for its functions and, in turn, for neural disorders. In this review, we discuss the roles of cadherins and related molecules in neural development and function in the vertebrate brain.


Question: Microarray outcomes from culture-amplified blends of serotypes hardly match with microarray outcomes from non-cultured blends.

The PneuCarriage Initiative: A Comprehensive Multi-Center Evaluation to Determine the Optimal Serotyping Techniques for Assessing Pneumococcal Carriage in Vaccine Research

CONTEXT The pneumococcus is a versatile pathogen predominantly found in the nasopharynx, with over 90 distinct serotypes. Concurrent nasopharyngeal carriage of multiple serotypes is frequent. To evaluate the efficacy of pneumococcal vaccines, a thorough understanding of pneumococcal carriage is crucial. Traditional serotyping techniques are laborious and often inadequate for identifying multiple serotype carriage. Moreover, there is a scarcity of comparative data regarding the newer serotyping methods developed in recent years. To address this gap, the PneuCarriage initiative was launched as a large-scale, international, multi-center study aimed at identifying the most effective pneumococcal serotyping techniques for carriage studies.

PROCEDURES AND OUTCOMES A set of reference samples was distributed to 15 research teams for blinded analysis. Twenty pneumococcal serotyping methods were applied to 81 laboratory-prepared (spiked) samples. The five best-performing methods were then used to analyze 260 nasopharyngeal (field) samples collected from children in six countries with high disease burden. The sensitivity and positive predictive value (PPV) were calculated for the tested methods and the reference method (traditional serotyping of >100 colonies from each sample). The sensitivity of the alternative serotyping methods ranged from 1% to 99% (reference method 98%), and the PPV from 8% to 100% (reference method 100%), when analyzing the spiked samples. Fifteen methods achieved ≥70% sensitivity for detecting the dominant (major) serotype, while only eight methods reached ≥70% sensitivity for identifying minor serotypes. For the field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method 93.8%), and the PPV from 82.2% to 96.4% (reference method 99.6%). The microarray, with a culture amplification step, demonstrated the highest sensitivity (95.8%) and a high PPV (93.7%). A key limitation of this study is that not all available alternative serotyping methods were included.

SUMMARY Most serotyping methods were effective in detecting the dominant serotype in a sample, but

Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used "survival" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.

COHCAP (City of Hope CpG Island Analysis Pipeline) is an algorithm to analyze single-nucleotide resolution DNA methylation data produced by either an Illumina methylation array or targeted bisulfite sequencing. The goal of the COHCAP algorithm is to identify CpG islands that show a consistent pattern of methylation among CpG sites. COHCAP is currently the only DNA methylation package that provides integration with gene expression data to identify a subset of CpG islands that are most likely to regulate downstream gene expression, and it can generate lists of differentially methylated CpG islands with ∼50% concordance with gene expression from both cell line data and heterogeneous patient data. For example, this article describes known breast cancer biomarkers (such as estrogen receptor) with a negative correlation between DNA methylation and gene expression. COHCAP also provides visualization for quality control metrics, regions of differential methylation and correlation between methylation and gene expression. This software is freely available at https://sourceforge.net/projects/cohcap/.

Background  Current criteria for the selection of unrelated donors for hematopoietic cell transplantation (HCT) include matching for the alleles of each human leukocyte antigen (HLA) locus within the major histocompatibility complex (MHC). Graft-versus-host disease (GVHD), however, remains a significant and potentially life-threatening complication even after HLA-identical unrelated HCT. The MHC harbors more than 400 genes, but the total number of transplantation antigens is unknown. Genes that influence transplantation outcome could be identified by using linkage disequilibrium (LD)-mapping approaches, if the extended MHC haplotypes of the unrelated donor and recipient could be defined.

BACKGROUND The annual number of hospital admissions and in-hospital deaths due to severe acute lower respiratory infections (ALRI) in young children worldwide is unknown. We aimed to estimate the incidence of admissions and deaths for such infections in children younger than 5 years in 2010. METHODS We estimated the incidence of admissions for severe and very severe ALRI in children younger than 5 years, stratified by age and region, with data from a systematic review of studies published between Jan 1, 1990, and March 31, 2012, and from 28 unpublished population-based studies. We applied these incidence estimates to population estimates for 2010, to calculate the global and regional burden in children admitted with severe ALRI in that year. We estimated in-hospital mortality due to severe and very severe ALRI by combining incidence estimates with case fatality ratios from hospital-based studies. FINDINGS We identified 89 eligible studies and estimated that in 2010, 11·9 million (95% CI 10·3-13·9 million) episodes of severe and 3·0 million (2·1-4·2 million) episodes of very severe ALRI resulted in hospital admissions in young children worldwide. Incidence was higher in boys than in girls, the sex disparity being greatest in South Asian studies. On the basis of data from 37 hospital studies reporting case fatality ratios for severe ALRI, we estimated that roughly 265,000 (95% CI 160,000-450,000) in-hospital deaths took place in young children, with 99% of these deaths in developing countries. Therefore, the data suggest that although 62% of children with severe ALRI are treated in hospitals, 81% of deaths happen outside hospitals. INTERPRETATION Severe ALRI is a substantial burden on health services worldwide and a major cause of hospital referral and admission in young children. Improved hospital access and reduced inequities, such as those related to sex and rural status, could substantially decrease mortality related to such infection. Community-based management of severe disease could be an important complementary strategy to reduce pneumonia mortality and health inequities. FUNDING WHO.

Nonreplicating and metabolically quiescent bacteria are implicated in latent tuberculosis infections and relapses following "sterilizing" chemotherapy. However, evidence linking bacterial dormancy and persistence in vivo is largely inconclusive. Here we measure the single-cell dynamics of Mycobacterium tuberculosis replication and ribosomal activity using quantitative time-lapse microscopy and a reporter of ribosomal RNA gene expression. Single-cell dynamics exhibit heterogeneity under standard growth conditions, which is amplified by stressful conditions such as nutrient limitation, stationary phase, intracellular replication, and growth in mouse lungs. Additionally, the lungs of chronically infected mice harbor a subpopulation of nongrowing but metabolically active bacteria, which are absent in mice lacking interferon-γ, a cytokine essential for antituberculosis immunity. These cryptic bacterial forms are prominent in mice treated with the antituberculosis drug isoniazid, suggesting a role in postchemotherapeutic relapses. Thus, amplification of bacterial phenotypic heterogeneity in response to host immunity and drug pressure may contribute to tuberculosis persistence.

Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other immunological disorders.

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.

A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called "quantitative temporal viromics" (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

Respiratory epithelial cells play an active part in the host response to respiratory pathogens, such as Streptococcus pneumoniae, by releasing chemokines responsible for neutrophil recruitment. In order to investigate the role of specific pneumococcal virulence factors in eliciting CXC chemokine responses, type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562) were infected with S. pneumoniae D39 or mutants lacking choline-binding protein A (CbpA), pneumococcal surface protein A (PspA), or specific domains thereof. In response to wild-type D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA and interleukin-8 protein. This response was increased twofold when a cbpA deletion mutant (DeltaCbpA) was used, suggesting that CbpA inhibits CXC chemokine induction. All three N-terminal domains of CbpA are required for this effect, as in-frame deletion of the respective region of cbpA had the same effect on the CXC chemokine response as deletion of cbpA altogether. Infection with a pspA deletion mutant (DeltaPspA) led to a twofold decrease in the CXC chemokine response of A549 but not Detroit-562 cells, compared to infection with D39 at 2 h. Thus, PspA appears to have the ability to stimulate early CXC chemokine release from A549 cells. Deletion of the region of pspA encoding the first N-terminal alpha-helical domain reduced the ability of S. pneumoniae to elicit a chemokine response to the same degree as deletion of pspA altogether. Thus, the N termini of CbpA and PspA exert differential effects on CXC chemokine induction in epithelial cells infected with S. pneumoniae.

Despite advances in medical device fabrication and antimicrobial treatment therapies, fungal-bacterial polymicrobial peritonitis remains a serious complication for surgery patients, those on peritoneal dialysis, and the critically ill. Using a murine model of peritonitis, we have demonstrated that monomicrobial infection with Candida albicans or Staphylococcus aureus is nonlethal. However, coinfection with these same doses leads to a 40% mortality rate and increased microbial burden in the spleen and kidney by day 1 postinfection. Using a multiplex enzyme-linked immunosorbent assay, we have also identified a unique subset of innate proinflammatory cytokines (interleukin-6, granulocyte colony-stimulating factor, keratinocyte chemoattractant, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α) that are significantly increased during polymicrobial versus monomicrobial peritonitis, leading to increased inflammatory infiltrate into the peritoneum and target organs. Treatment of coinfected mice with the cyclooxygenase (COX) inhibitor indomethacin reduces the infectious burden, proinflammatory cytokine production, and inflammatory infiltrate while simultaneously preventing any mortality. Further experiments demonstrated that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) is synergistically increased during coinfection compared to monomicrobial infection; indomethacin treatment also decreased elevated PGE2 levels. Furthermore, addition of exogenous PGE2 into the peritoneal cavity during infection overrode the protection provided by indomethacin and restored the increased mortality and microbial burden. Importantly, these studies highlight the ability of fungal-bacterial coinfection to modulate innate inflammatory events with devastating consequences to the host.


Question: Mitochondria are not involved in the process of apoptosis.

Mitochondria: Vital Organelles in Health, Disease, and Aging
Mitochondria serve as the powerhouse of most eukaryotic cells, generating energy through oxidative phosphorylation. Beyond their energy-producing role, mitochondria are involved in various critical cellular processes, including intermediary metabolism, calcium homeostasis, and programmed cell death, or apoptosis. Considering their multifaceted functions, mitochondrial impairment could theoretically lead to a uniform set of cellular deficiencies. Yet, in complex organisms, mitochondrial dysfunction manifests in diverse and unexpected ways, affecting multiple systems. It's evident that the fundamental aspects of mitochondrial biology are not yet fully elucidated. In this review, we highlight recent findings that underscore the significance of mitochondrial dynamics, specifically their fusion and fission cycles, in the context of development and disease pathology.

We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.

The SNF1/AMP-activated protein kinase (AMPK) family maintains the balance between ATP production and consumption in all eukaryotic cells. The kinases are heterotrimers that comprise a catalytic subunit and regulatory subunits that sense cellular energy levels. When energy status is compromised, the system activates catabolic pathways and switches off protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation. Surprisingly, recent results indicate that the AMPK system is also important in functions that go beyond the regulation of energy homeostasis, such as the maintenance of cell polarity in epithelial cells.

The important life-supporting role of hydrogen sulfide (H(2)S) has evolved from bacteria to plants, invertebrates, vertebrates, and finally to mammals. Over the centuries, however, H(2)S had only been known for its toxicity and environmental hazard. Physiological importance of H(2)S has been appreciated for about a decade. It started by the discovery of endogenous H(2)S production in mammalian cells and gained momentum by typifying this gasotransmitter with a variety of physiological functions. The H(2)S-catalyzing enzymes are differentially expressed in cardiovascular, neuronal, immune, renal, respiratory, gastrointestinal, reproductive, liver, and endocrine systems and affect the functions of these systems through the production of H(2)S. The physiological functions of H(2)S are mediated by different molecular targets, such as different ion channels and signaling proteins. Alternations of H(2)S metabolism lead to an array of pathological disturbances in the form of hypertension, atherosclerosis, heart failure, diabetes, cirrhosis, inflammation, sepsis, neurodegenerative disease, erectile dysfunction, and asthma, to name a few. Many new technologies have been developed to detect endogenous H(2)S production, and novel H(2)S-delivery compounds have been invented to aid therapeutic intervention of diseases related to abnormal H(2)S metabolism. While acknowledging the challenges ahead, research on H(2)S physiology and medicine is entering an exponential exploration era.

The average lifespan of humans is increasing, and with it the percentage of people entering the 65 and older age group is growing rapidly and will continue to do so in the next 20 years. Within this age group, cardiovascular disease will remain the leading cause of death, and the cost associated with treatment will continue to increase. Aging is an inevitable part of life and unfortunately poses the largest risk factor for cardiovascular disease. Although numerous studies in the cardiovascular field have considered both young and aged humans, there are still many unanswered questions as to how the genetic pathways that regulate aging in model organisms influence cardiovascular aging. Likewise, in the molecular biology of aging field, few studies fully assess the role of these aging pathways in cardiovascular health. Fortunately, this gap is beginning to close, and these two fields are merging together. We provide an overview of some of the key genes involved in regulating lifespan and health span, including sirtuins, AMP-activated protein kinase, mammalian target of rapamycin, and insulin-like growth factor 1 and their roles regulating cardiovascular health. We then discuss a series of review articles that will appear in succession and provide a more comprehensive analysis of studies carried out linking genes of aging and cardiovascular health, and perspectives of future directions of these two intimately linked fields.

Neutrophilic polymorphonuclear leukocytes (neutrophils) are highly specialized for their primary function, the phagocytosis and destruction of microorganisms. When coated with opsonins (generally complement and/or antibody), microorganisms bind to specific receptors on the surface of the phagocyte and invagination of the cell membrane occurs with the incorporation of the microorganism into an intracellular phagosome. There follows a burst of oxygen consumption, and much, if not all, of the extra oxygen consumed is converted to highly reactive oxygen species. In addition, the cytoplasmic granules discharge their contents into the phagosome, and death of the ingested microorganism soon follows. Among the antimicrobial systems formed in the phagosome is one consisting of myeloperoxidase (MPO), released into the phagosome during the degranulation process, hydrogen peroxide (H2O2), formed by the respiratory burst and a halide, particularly chloride. The initial product of the MPO-H2O2-chloride system is hypochlorous acid, and subsequent formation of chlorine, chloramines, hydroxyl radicals, singlet oxygen, and ozone has been proposed. These same toxic agents can be released to the outside of the cell, where they may attack normal tissue and thus contribute to the pathogenesis of disease. This review will consider the potential sources of H2O2 for the MPO-H2O2-halide system; the toxic products of the MPO system; the evidence for MPO involvement in the microbicidal activity of neutrophils; the involvement of MPO-independent antimicrobial systems; and the role of the MPO system in tissue injury. It is concluded that the MPO system plays an important role in the microbicidal activity of phagocytes.

Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd−/− cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.

Previous studies have detected associations between mitochondrial haplogroups and schizophrenia (SZ). However, no study has examined the relationship between major mitochondrial DNA (mtDNA) haplogroups and SZ in the Chinese population. The aim of this study was to assess the association between mtDNA haplogroups and SZ genesis in the Chinese Han population. We used a case-control study and sequenced the mtDNA hypervariable regions (HVR1, HVR2, and HVR3) in the Han population. We analyzed mtDNA haplogroups and HVR polymorphisms in 298 SZ patients and 298 controls. The haplotypes were classified into 10 major haplogroups: A, B, CZ, D, F, G, M, N, N9a, and R. Statistical analysis revealed that only N9a showed a nominally significant association with protection from SZ [1.68% vs. 6.38%, p=0.004, OR=0.251 (0.092-0.680); after adjustment for age and sex: p=0.006, OR=0.246 (0.090-0.669)]. Three HVR polymorphisms were found to be nominally significantly different between subjects with SZ and controls, and all except one (m.204T>C) are linked to the N9a haplogroup. Our results indicate that mtDNA haplogroup N9a might be a protective factor for SZ.

Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Survivin is a member of the chromosome passenger complex, which plays an important role in chromosome alignment, separation, and cytokinesis. Although survivin is required for the proliferation and survival of hematopoietic stem and progenitor cells, the extent to which it is necessary for endomitosis of megakaryocytes remains controversial. To determine whether survivin is required for polyploidization, we analyzed mice with a megakaryocyte-specific deletion. PF4-Cre/survivin(fl/fl) mice harbored normal platelet counts with megakaryocytes that reached ploidy states comparable with those of control littermates. The CD41(+) cells within these animals showed little excision but increased annexin V staining, implying that survivin is required for survival of megakaryocyte progenitors in vivo. In contrast, megakaryocytes in which survivin was excised ex vivo showed robust excision and an increased degree of polyploidization. These results demonstrate that survivin is necessary for survival of megakaryocyte progenitors, but is not required for polyploidization of committed megakaryocytes.


Question: Altering the epigenome within the brain impacts the typical human aging process by influencing specific genes associated with neurogenesis.

Epigenetic Regulation in Neurological Disorders and Aging
Recent studies into the complexities of the brain's epigenetic landscape, encompassing DNA methylation patterns and histone modifications, have unveiled novel perspectives on neural development, the etiology of neurological diseases, and the aging process. Historically, chromatin abnormalities in the brain were viewed as fixed developmental defects linked to rare genetic conditions. However, the latest findings indicate that mutations and dysfunctions in epigenetic processes span a broader spectrum, encompassing late-onset neurodegenerative diseases. This article reviews how the field of neuroepigenetics has deepened our understanding of the pathophysiology behind developmental and degenerative brain conditions. Furthermore, it contemplates the potential implications of these discoveries for the creation of innovative therapeutic strategies to combat neurological disorders.

Somatic stem cell depletion due to the accumulation of DNA damage has been implicated in the appearance of aging-related phenotypes. Hair graying, a typical sign of aging in mammals, is caused by the incomplete maintenance of melanocyte stem cells (MSCs) with age. Here, we report that irreparable DNA damage, as caused by ionizing radiation, abrogates renewal of MSCs in mice. Surprisingly, the DNA-damage response triggers MSC differentiation into mature melanocytes in the niche, rather than inducing their apoptosis or senescence. The resulting MSC depletion leads to irreversible hair graying. Furthermore, deficiency of Ataxia-telangiectasia mutated (ATM), a central transducer kinase of the DNA-damage response, sensitizes MSCs to ectopic differentiation, demonstrating that the kinase protects MSCs from their premature differentiation by functioning as a "stemness checkpoint" to maintain the stem cell quality and quantity.

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.

Stem cells persist throughout life in diverse tissues by undergoing self-renewing divisions. Self-renewal capacity declines with age, partly because of increasing expression of the tumor suppressor p16(Ink4a). We discovered that the Hmga2 transcriptional regulator is highly expressed in fetal neural stem cells but that expression declines with age. This decrease is partly caused by the increasing expression of let-7b microRNA, which is known to target HMGA2. Hmga2-deficient mice show reduced stem cell numbers and self-renewal throughout the central and peripheral nervous systems of fetal and young-adult mice but not old-adult mice. Furthermore, p16(Ink4a) and p19(Arf) expression were increased in Hmga2-deficient fetal and young-adult stem cells, and deletion of p16(Ink4a) and/or p19(Arf) partially restored self-renewal capacity. let-7b overexpression reduced Hmga2 and increased p16(Ink4a)/p19(Arf) expression. Hmga2 thus promotes fetal and young-adult stem cell self-renewal by decreasing p16(Ink4a)/p19(Arf) expression. Changes in let-7 and Hmga2 expression during aging contribute to the decline in neural stem cell function.

Background: Reduced brain insulin signaling and low CSF-to-plasma insulin ratios have been observed in patients with Alzheimer disease (AD). Furthermore, intracerebroventricular or IV insulin administration improve memory, alter evoked potentials, and modulate neurotransmitters, possibly by augmenting low brain levels. After intranasal administration, insulin-like peptides follow extracellular pathways to the brain within 15 minutes. Objective: We tested the hypothesis that daily intranasal insulin treatment would facilitate cognition in patients with early AD or its prodrome, amnestic mild cognitive impairment (MCI). The proportion of verbal information retained after a delay period was the planned primary outcome measure. Secondary outcome measures included attention, caregiver rating of functional status, and plasma levels of insulin, glucose, β-amyloid, and cortisol. Methods: Twenty-five participants were randomly assigned to receive either placebo (n = 12) or 20 IU BID intranasal insulin treatment (n = 13) using an electronic atomizer, and 24 participants completed the study. Participants, caregivers, and all clinical evaluators were blinded to treatment assignment. Cognitive measures and blood were obtained at baseline and after 21 days of treatment. Results: Fasting plasma glucose and insulin were unchanged with treatment. The insulin-treated group retained more verbal information after a delay compared with the placebo-assigned group ( p = 0.0374). Insulin-treated subjects also showed improved attention ( p = 0.0108) and functional status ( p = 0.0410). Insulin treatment raised fasting plasma concentrations of the short form of the β-amyloid peptide (Aβ40; p = 0.0471) without affecting the longer isoform (Aβ42), resulting in an increased Aβ40/42 ratio ( p = 0.0207). Conclusions: The results of this pilot study support further investigation of the benefits of intranasal insulin for patients with Alzheimer disease, and suggest that intranasal peptide administration may be a novel approach to the treatment of neurodegenerative disorders.

Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.

Genome-wide DNA methylation reprogramming occurs in mouse primordial germ cells (PGCs) and preimplantation embryos, but the precise dynamics and biological outcomes are largely unknown. We have carried out whole-genome bisulfite sequencing (BS-Seq) and RNA-Seq across key stages from E6.5 epiblast to E16.5 PGCs. Global loss of methylation takes place during PGC expansion and migration with evidence for passive demethylation, but sequences that carry long-term epigenetic memory (imprints, CpG islands on the X chromosome, germline-specific genes) only become demethylated upon entry of PGCs into the gonads. The transcriptional profile of PGCs is tightly controlled despite global hypomethylation, with transient expression of the pluripotency network, suggesting that reprogramming and pluripotency are inextricably linked. Our results provide a framework for the understanding of the epigenetic ground state of pluripotency in the germline.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a , profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age-related bone loss. © 2016 American Society for Bone and Mineral Research.

Longevity is regulated by the daf-2 gene network in Caenorhabditis elegans. Mutations in the daf-2 gene, which encodes a member of the insulin receptor family, confer the life extension (Age) phenotype and the constitutive dauer (a growth-arrested larval form specialized for dispersal) formation phenotype. The Age phenotype is mutually potentiated by two life extension mutations in the daf-2 gene and the clk-1 gene, a homologue of yeast CAT5/COQ7 known to regulate ubiquinone biosynthesis. In this study, we demonstrated that the daf-2 mutation also conferred an oxidative stress resistance (Oxr) phenotype, which was also enhanced by the clk-1 mutation. Similar to the Age phenotype, the Oxr phenotype was regulated by the genetic pathway of insulin-like signaling from daf-2 to the daf-16 gene, a homologue of the HNF-3/forkhead transcription factor. These findings led us to examine whether the insulin-like signaling pathway regulates the gene expression of antioxidant defense enzymes. We found that the mRNA level of the sod-3 gene, which encodes Mn-superoxide dismutase (SOD), was much higher in daf-2 mutants than in the wild type. Moreover, the increased sod-3 gene expression phenotype is regulated by the insulin-like signaling pathway. Although the clk-1 mutant itself did not display Oxr and the increased sod-3 expression phenotypes, the clk-1 mutation enhanced them in the daf-2 mutant, suggesting that clk-1 is involved in longevity in two ways: clk-1 composes the original clk-1 longevity program and the daf-2 longevity program. These observations suggest that the daf-2 gene network controls longevity by regulating the Mn-SOD-associated antioxidant defense system. This system appears to play a role in efficient life maintenance at the dauer stage.

Control of translation is a fundamental source of regulation in gene expression. The induction of protein synthesis by brain-derived neurotrophic factor (BDNF) critically contributes to enduring modifications of synaptic function, but how BDNF selectively affects only a minority of expressed mRNAs is poorly understood. We report that BDNF rapidly elevates Dicer, increasing mature miRNA levels and inducing RNA processing bodies in neurons. BDNF also rapidly induces Lin28, causing selective loss of Lin28-regulated miRNAs and a corresponding upregulation in translation of their target mRNAs. Binding sites for Lin28-regulated miRNAs are necessary and sufficient to confer BDNF responsiveness to a transcript. Lin28 deficiency, or expression of a Lin28-resistant Let-7 precursor miRNA, inhibits BDNF translation specificity and BDNF-dependent dendrite arborization. Our data establish that specificity in BDNF-regulated translation depends upon a two-part posttranscriptional control of miRNA biogenesis that generally enhances mRNA repression in association with GW182 while selectively derepressing and increasing translation of specific mRNAs.

Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1/Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's life may be partially explained by a miRNA-directed mechanism of age-associated decline.


Question: Monoclonal antibody directed at N-cadherin prevents metastasis.

Targeting N-cadherin with Monoclonal Antibodies: A Promising Strategy to Combat Prostate Cancer Metastasis and Castration Resistance. The progression from androgen-sensitive to castration-resistant prostate cancer (CRPC) represents a critical, often fatal, transition with unclear molecular underpinnings. Through comparative gene expression analysis between isogenic androgen-dependent and CRPC xenograft models, we observed a consistent upregulation of N-cadherin, a finding corroborated in primary and metastatic tumors from patients with CRPC. Forced expression of N-cadherin in nonmetastatic, androgen-sensitive prostate cancer models triggered castration resistance, enhanced invasiveness, and promoted metastasis. Treatment with monoclonal antibodies directed against the extracellular domain of N-cadherin significantly curtailed the proliferation, adhesion, and invasive capabilities of prostate cancer cells in vitro. In vivo studies revealed that these antibodies effectively retarded the growth of established CRPC xenografts, inhibited local invasion and metastasis, and at elevated dosages, induced tumor regression. Importantly, N-cadherin-targeting antibodies substantially prolonged the onset of castration resistance, altered tumor architecture and angiogenesis, and diminished AKT kinase activity and serum interleukin-8 (IL-8) levels. Collectively, these findings underscore the pivotal role of N-cadherin in prostate cancer metastasis and castration resistance, suggesting that monoclonal antibody therapy targeting this molecule could yield significant therapeutic benefits in clinical settings.

Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK–ERK–MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer.

Small non-coding microRNAs (miRNAs) contribute to cancer development and progression, and are differentially expressed in normal tissues and cancers. However, the specific role of miRNAs in the metastatic process is still unknown. To seek a specific miRNA expression signature characterizing the metastatic phenotype of solid tumours, we performed a miRNA microarray analysis on 43 paired primary tumours (ten colon, ten bladder, 13 breast, and ten lung cancers) and one of their related metastatic lymph nodes. We identified a metastatic cancer miRNA signature comprising 15 overexpressed and 17 underexpressed miRNAs. Our results were confirmed by qRT-PCR analysis. Among the miRNAs identified, some have a well-characterized association with cancer progression, eg miR-10b, miR-21, miR-30a, miR-30e, miR-125b, miR-141, miR-200b, miR-200c, and miR-205. To further support our data, we performed an immunohistochemical analysis for three well-defined miRNA gene targets (PDCD4, DHFR, and HOXD10 genes) on a small series of paired colon, breast, and bladder cancers, and one of their metastatic lymph nodes. We found that the immunohistochemical expression of these targets significantly follows the corresponding miRNA deregulation. Our results suggest that specific miRNAs may be directly involved in cancer metastasis and that they may represent a novel diagnostic tool in the characterization of metastatic cancer gene targets.

Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test—and readily amenable to serial sampling—have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch® system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial–mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.

Atherosclerotic plaques develop in regions of the vasculature associated with chronic inflammation due to disturbed flow patterns. Endothelial phenotype modulation by flow requires the integration of numerous mechanotransduction pathways, but how this is achieved is not well understood. We show here that, in response to flow, the adaptor protein Shc is activated and associates with cell-cell and cell-matrix adhesions. Shc activation requires the tyrosine kinases vascular endothelial growth factor receptor 2 and Src. Shc activation and its vascular endothelial cadherin (VE-cadherin) association are matrix independent. In contrast, Shc binding to integrins requires VE-cadherin but occurs only on specific matrices. Silencing Shc results in reduction in both matrix-independent and matrix-dependent signals. Furthermore, Shc regulates flow-induced inflammatory signaling by activating nuclear factor kappaB-dependent signals that lead to atherogenesis. In vivo, Shc is activated in atherosclerosis-prone regions of arteries, and its activation correlates with areas of atherosclerosis. Our results support a model in which Shc orchestrates signals from cell-cell and cell-matrix adhesions to elicit flow-induced inflammatory signaling.

Mouse ovarian surface epithelial cells (MOSEC) were obtained from virgin, mature mice by mild trypsinization and were repeatedly passaged in vitro. Early passage cells (<20 passages) exhibited a cobblestone morphology and contact inhibition of growth. After approximately 20 passages in vitro, cobblestone morphology and contact inhibition of growth was lost. Tumor forming potential was determined by s.c. and i.p. injection of early and late passage cells into athymic and syngeneic C57BL6 mice. Subcutaneous tumors formed in approximately 4 months and were present only at the injection site. Intraperitoneal injection of late passage MOSEC into athymic and syngeneic mice resulted in growth of tumor implants throughout the abdominal cavity, and production of hemorrhagic ascitic fluid. Early passage MOSEC did not form tumors in vivo. Histopathologic analysis of tumors revealed a highly malignant neoplasm containing both carcinomatous and sarcomatous components. Late passage MOSEC expressed cytokeratin and did not produce ovarian steroids in response to gonadotropin stimulation in vitro. Ten clonal lines were established from late passage MOSEC. Each clone formed multiple peritoneal tumors and ascitic fluid after i.p. injection into C57BL6 mice. Three cell lines examined cytogenetically were polyploid with near-tetraploid modal chromosome numbers. Common clonal chromosome gains and losses included +5, +15, +19 and -X, -3, -4. One cell line had a clonal translocation between chromosomes 15 and 18 and another had a small marker chromosome; common structural abnormalities were not observed. These data describe the development of a mouse model for the study of events related to ovarian cancer in humans. The ability of the MOSEC to form extensive tumors within the peritoneal cavity, similar to those seen in women with Stage III and IV cancer, and the ability of the MOSEC to produce tumors in mice with intact immune systems, makes this model unique for investigations of molecular and immune interactions in ovarian cancer development.

S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

BACKGROUND Although zoledronic acid (ZOL), a third-generation nitrogen-containing bisphosphonate, has been identified as an attractive therapeutic agent against breast cancer, prostate cancer, multiple myeloma as well as small-cell lung cancer (SCLC), as best as we are aware, the anti-tumor effect of ZOL upon non-small-cell lung cancer (NSCLC) remains to be effectively investigated. This study examined the effects of ZOL upon the line-1 tumor cell, using a murine lung adenocarcinoma cell line similar to the behavior of human lung adenocarcinoma. METHODS We investigated the anti-tumor effects of ZOL (3-100 microM) on line-1 tumor cells in vitro, including cellular proliferation, by means of an MTT assay, cell-cycle analysis by flow cytometry and by assessing the level of apoptosis by annexin V/propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) staining. Further, we evaluated the growth and survival of line-1 tumor cells following ZOL treatment (1 microg/kg/week) using an animal model. We also examined the in vivo cell-cycle pattern using lacZ-expressing line-1 cells (line-1/lacZ). RESULTS ZOL significantly slowed the line-1 tumor growth in a dose-dependent manner in vitro. The treated line-1 tumor cells typically arrested at the S/G2/M-phase of the cell-cycle following ZOL exposure, but no apoptotic cells could be detected by either annexin V/PI or DAPI staining. When the ZOL was washed out, the drug-inhibited cells continued to proliferate again and the cell-cycle prolongation elicited earlier by the drug, then disappeared. Within 72-96 h following drug removal, the cell-cycle of the treated cells revealed a similar distribution to that of the untreated controls. In vivo studies demonstrated that ZOL significantly slowed the line-1 tumor growth. Indeed, mice lived significantly longer when they had been ZOL-treated than was the case for untreated mice (p<0.05). Using line-1/lacZ cells, the in vivo cell-cycle distribution of line-1 tumor cells subsequent to ZOL exposure revealed S/G2/M-phase arrest that was identical to the in vitro culture. CONCLUSIONS ZOL maintains the potential to reduce tumor burden and prolong survival for murine pulmonary adenocarcinoma. The flow cytometrical analysis of cell-cycle demonstrated that ZOL induces no apoptosis but is able to arrest line-1 tumor cells at the S/G2/M-phase. Although the clinical relevance of these results warrants verification for human lung cancer patients, ZOL combined with chemotherapy and/or radiotherapy appears to be a new therapeutic strategy for the effective treatment of NSCLC.

New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. Hence, the molecular basis of tumor angiogenesis has been of keen interest in the field of cancer research. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process. The VEGF/VEGF-receptor axis is composed of multiple ligands and receptors with overlapping and distinct ligand-receptor binding specificities, cell-type expression, and function. Activation of the VEGF-receptor pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. In addition, VEGF mediates vessel permeability, and has been associated with malignant effusions. More recently, an important role for VEGF has emerged in mobilization of endothelial progenitor cells from the bone marrow to distant sites of neovascularization. The well-established role of VEGF in promoting tumor angiogenesis and the pathogenesis of human cancers has led to the rational design and development of agents that selectively target this pathway. Studies with various anti-VEGF/VEGF-receptor therapies have shown that these agents can potently inhibit angiogenesis and tumor growth in preclinical models. Recently, an anti-VEGF antibody (bevacizumab), when used in combination with chemotherapy, was shown to significantly improve survival and response rates in patients with metastatic colorectal cancer and thus, validate VEGF pathway inhibitors as an important new treatment modality in cancer therapy.

The present study was designed to determine the effects of physical training on the development of cancer induced by the injection of Ehrlich tumor cells in mice. Male Swiss mice were subjected to a swim training protocol (5 days/wk for 6 wk, 1 h at 50% of maximal capacity-trained groups) or remained sedentary in their cages (sedentary groups). The inoculation of Ehrlich tumor cells was performed at the end of the fourth week, and animals were killed after 6 wk of training. Heart and solid tumor weights were recorded, and tumor volumes were calculated. Portions of the tumors were used for the evaluation of macrophages and neutrophil accumulation or fixed in neutral 10% buffered formalin for histological analysis. The tumor volume and weight were, respectively, approximately 270% and 280% greater in sedentary mice than in trained mice. Macrophage infiltration in the tumor tissue was significantly lower in trained mice (0.65 +/- 0.16 vs. 1.78 +/- 0.43 macrophages x 10(3) in the sedentary group). Moreover, neutrophil accumulation in tumors was slightly reduced after exercise training, and the amount of tumor cells was reduced in trained mice. Exercise capacity was substantially increased in trained mice, as determined by a 440% increase in the exercise time at 50% of maximal capacity. In summary, swim training retarded the development of Ehrlich tumors in mice, accompanied by a reduction in macrophage infiltration and neutrophil accumulation. These findings provide conceptual support for clinical observations that controlled physical activities may be a therapeutically important approach to preventing cancer progression and may improve the outcome of cancer treatment.


Question: The majority of termination instances in Okazaki fragments are sequence-specific.

Comprehensive Genome-wide Investigation of Eukaryotic DNA Replication Dynamics.

Despite considerable progress in the field of molecular biology, several key aspects of DNA replication remain elusive, including the precise sites of initiation and termination, the frequency of origin usage, and the impact of transcription on fork progression. By employing the deep sequencing of Okazaki fragments, we have generated a detailed map of replication fork directionality across the entire S. cerevisiae genome. This extensive dataset enables us to conduct a systematic examination of replication initiation, origin efficiency, fork movement, and termination dynamics.

Our findings reveal that the initiation of leading-strand synthesis is primarily confined to nucleosome-free regions at replication origins. Through the use of a strain where late origins can be prematurely activated, we have determined that replication termination is predominantly a passive process that does not depend on specific DNA sequences or stalling of replication forks. Instead, the replication profile is chiefly influenced by the timing of origin firing, which allows us to accurately predict chromosome-wide replication timing from an asynchronous culture.

These insights into the mechanisms underlying DNA replication provide a solid foundation for further research into the complex interplay between replication and other cellular processes.

By employing the nuclear DNA of the African rice Oryza glaberrima as a reference genome, the timing, natures, mechanisms, and specificities of recent sequence evolution in the indica and japonica subspecies of Oryza sativa were identified. The data indicate that the genome sizes of both indica and japonica have increased substantially, >2% and >6%, respectively, since their divergence from a common ancestor, mainly because of the amplification of LTR-retrotransposons. However, losses of all classes of DNA sequence through unequal homologous recombination and illegitimate recombination have attenuated the growth of the rice genome. Small deletions have been particularly frequent throughout the genome. In >1 Mb of orthologous regions that we analyzed, no cases of complete gene acquisition or loss from either indica or japonica were found, nor was any example of precise transposon excision detected. The sequences between genes were observed to have a very high rate of divergence, indicating a molecular clock for transposable elements that is at least 2-fold more rapid than synonymous base substitutions within genes. We found that regions prone to frequent insertions and deletions also exhibit higher levels of point mutation. These results indicate a highly dynamic rice genome with competing processes for the generation and removal of genetic variation.

G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.

TAL (transcription activator-like) effectors constitute a novel class of DNA-binding proteins with predictable specificity. They are employed by Gram-negative plant-pathogenic bacteria of the genus Xanthomonas which translocate a cocktail of different effector proteins via a type III secretion system (T3SS) into plant cells where they serve as virulence determinants. Inside the plant cell, TALs localize to the nucleus, bind to target promoters, and induce expression of plant genes. DNA-binding specificity of TALs is determined by a central domain of tandem repeats. Each repeat confers recognition of one base pair (bp) in the DNA. Rearrangement of repeat modules allows design of proteins with desired DNA-binding specificities. Here, we summarize how TAL specificity is encoded, first structural data and first data on site-specific TAL nucleases.

During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts-Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe-also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10(-8), which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. Replication forks stall at CGG repeats in a length-dependent manner in primate cells and in yeast. Saccharomyces cerevisiae proteins Tof1 and Mrc1 facilitate replication fork progression through CGG repeats. Remarkably, the fork-stabilizing role of Mrc1 does not involve its checkpoint function. Thus, chromosomal fragility might occur when forks stalled at expanded CGG repeats escape the S-phase checkpoint.

MOTIVATION Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. RESULTS The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. AVAILABILITY AND IMPLEMENTATION Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic   CONTACT usadel@bio1.rwth-aachen.de   SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Homothallic yeast cells undergo a specific pattern of mating-type switching initiated by an endonuclease encoded by the HO gene. HO transcription is affected by cell type (a, alpha, and a/alpha), by cell age (mother or daughter), and by the cell cycle. This paper investigates the sequences involved in HO transcription by replacing genomic DNA with copies mutated in vitro. A region between -1000 and 1400 (called URS1) is necessary for transcription in addition to a "TATA"-like region at -90. The 900 bp of DNA separating URS1 from the "TATA" box is not necessary for transcription nor for a/alpha repression and some measure of mother/daughter control, but it is necessary for correct cell cycle control.

RuvBL1 is an evolutionarily highly conserved eukaryotic protein belonging to the AAA(+)-family of ATPases (ATPase associated with diverse cellular activities). It plays important roles in essential signaling pathways such as the c-Myc and Wnt pathways in chromatin remodeling, transcriptional and developmental regulation, and DNA repair and apoptosis. Herein we present the three-dimensional structure of the selenomethionine variant of human RuvBL1 refined using diffraction data to 2.2A of resolution. The crystal structure of the hexamer is formed of ADP-bound RuvBL1 monomers. The monomers contain three domains, of which the first and the third are involved in ATP binding and hydrolysis. Although it has been shown that ATPase activity of RuvBL1 is needed for several in vivo functions, we could only detect a marginal activity with the purified protein. Structural homology and DNA binding studies demonstrate that the second domain, which is unique among AAA(+) proteins and not present in the bacterial homolog RuvB, is a novel DNA/RNA-binding domain. We were able to demonstrate that RuvBL1 interacted with single-stranded DNA/RNA and double-stranded DNA. The structure of the RuvBL1.ADP complex, combined with our biochemical results, suggest that although RuvBL1 has all the structural characteristics of a molecular motor, even of an ATP-driven helicase, one or more as yet undetermined cofactors are needed for its enzymatic activity.

Telomeres resemble common fragile sites (CFSs) in that they are difficult-to-replicate and exhibit fragility in mitosis in response to DNA replication stress. At CFSs, this fragility is associated with a delay in the completion of DNA replication until early mitosis, whereupon cells are proposed to switch to a RAD52-dependent form of break-induced replication. Here, we show that this mitotic DNA synthesis (MiDAS) is also a feature of human telomeres. Telomeric MiDAS is not restricted to those telomeres displaying overt fragility, and is a feature of a wide range of cell lines irrespective of whether their telomeres are maintained by telomerase or by the alternative lengthening of telomeres (ALT) mechanism. MiDAS at telomeres requires RAD52, and is mechanistically similar to CFS-associated MiDAS, with the notable exception that telomeric MiDAS does not require the MUS81-EME1 endonuclease. We propose a model whereby replication stress initiates a RAD52-dependent form of break-induced replication that bypasses a requirement for MUS81-EME1 to complete DNA synthesis in mitosis.