Question: The single flash-evoked ERG b-wave is generated by activity of ON-bipolar cells.

Title: A naturally occurring mouse model of X-linked congenital stationary night blindness. Content: PURPOSE To describe a naturally occurring X-linked recessive mutation, no b-wave (nob), that compromises visual transmission between photoreceptors and second-order neurons in mice. METHODS Affected mice were identified by recording the light-evoked response of the retina, the electroretinogram (ERG). To evaluate visual transmission, cortical potentials were recorded with a scalp electrode. The inheritance pattern for nob was defined by breeding nob animals with normal mice. Retinal histologic analysis was performed by light microscopy. RESULTS Although the photoreceptor-mediated ERG component (a-wave) was normal in nob mice, the major response component reflecting postreceptoral neuronal activity (b-wave) was missing. Visually-driven cortical activity was also abnormal in nob animals. At the light microscopic level, the nob retina appeared to have a normal cytoarchitecture. CONCLUSIONS These findings suggest that the nob defect interferes with the transmission of visual information through the retina and that these mice are a useful model for the study of outer retinal synaptic function. In addition, this mutant mouse seems to provide an animal model for the complete form of congenital stationary night blindness, a human disorder in which patients have a profound loss of rod-mediated visual sensitivity.

Title: The reclamation of brine generated from desalination process by bipolar membrane electrodialysis Content: Abstract Some techno-economical analyses and environmental impact evaluation have proved that bipolar membrane electrodialysis (BMED) is feasible for the reclamation of industrial saline water. However, to date, the technology cannot be put into practice due to some unsolved application-oriented problems, such as the requirements of BMED for feed solution, the availability of the produced acid and base, relatively high salt concentration of the effluent, and relatively low desalinating efficiency and capacity. In this work, a novel hybrid process, which coupled conventional electrodialysis installed with monovalent selective cation-exchange membranes with BMED running in a constant-voltage mode, was designed to reclaim brine generated from surface water desalination by the ion-exchange process. Subsequently, the response surface methodology was employed to establish the empirical models for understanding the influences of some initial operating conditions on BMED performance. Finally, the BMED-based reclamation scheme was confirmed again by a continuous BMED experiment on real solution. Specially, the effects of product concentration on current efficiency and energy consumption were investigated. In this case, an acceptable current efficiency and energy consumption were obtained on the basis of the conventional membranes and spacers when the product concentration was set as 0.9 M, which is adequate for the regeneration of ion-exchange resins.

Title: Autocrine BDNF–TrkB signalling within a single dendritic spine Content: Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity, including structural long-term potentiation (sLTP), which is a correlate of an animal’s learning. However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where. Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy, we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices. In response to sLTP induction, we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR (N-methyl-d-aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF. We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons. Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin. We demonstrate that this postsynaptic BDNF–TrkB signalling pathway is necessary for both structural and functional LTP. Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR–CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.

Title: Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge. Content: Slow changes in [Ca(2+)](i) reflect increased neuronal activity. Our study demonstrates that single-trial fast [Ca(2+)](i) imaging (≥200 Hz sampling rate) revealed peaks each of which are associated with single spike discharge recorded by consecutive voltage-sensitive dye (VSD) imaging in enteric neurones and nerve fibres. Fast [Ca(2+)](i) imaging also revealed subthreshold fast excitatory postsynaptic potentials. Nicotine-evoked [Ca(2+)](i) peaks were reduced by -conotoxin and blocked by ruthenium red or tetrodotoxin. Fast [Ca(2+)](i) imaging can be used to directly record single action potentials in enteric neurones. [Ca(2+)](i) peaks required opening of voltage-gated sodium and calcium channels as well as Ca(2+) release from intracellular stores.

Title: Increased CDK1 activity determines the timing of kinetochore-microtubule attachments in meiosis I Content: Chromosome segregation during cell division depends on stable attachment of kinetochores to spindle microtubules. Mitotic spindle formation and kinetochore-microtubule (K-MT) capture typically occur within minutes of nuclear envelope breakdown. In contrast, during meiosis I in mouse oocytes, formation of the acentrosomal bipolar spindle takes 3-4 h, and stabilization of K-MT attachments is delayed an additional 3-4 h. The mechanism responsible for this delay, which likely prevents stabilization of erroneous attachments during spindle formation, is unknown. Here we show that during meiosis I, attachments are regulated by CDK1 activity, which gradually increases through prometaphase and metaphase I. Partial reduction of CDK1 activity delayed formation of stable attachments, whereas a premature increase in CDK1 activity led to precocious formation of stable attachments and eventually lagging chromosomes at anaphase I. These results indicate that the slow increase in CDK1 activity in meiosis I acts as a timing mechanism to allow stable K-MT attachments only after bipolar spindle formation, thus preventing attachment errors.

Title: Surfactant Protein B Suppresses Lung Cancer Progression by Inhibiting Secretory Phospholipase A2 Activity and Arachidonic Acid Production Content: Background/Aims: Radiotherapy is applied to patients with inoperable cancer types including advanced stage non-small cell lung cancer (NSCLC) and radioresistance functions as a critical obstacle in radiotherapy. This study was aimed to investigate the mechanism of radioresistance regulated by surfactant protein B (SP-B). Methods: To investigate the role of SP-B in radioresistance, ΔSFTPB A549 cell line was established and SP-B expression was analyzed. In response to ionizing radiation (IR), the change of SP-B expression was analyzed in A549 and NCI-H441 cell lines. Conditioned media (CM) from NSCLC cells were utilized to evaluate the downstream signaling pathway. The in vivo effects of SP-B were assessed through mouse xenograft model with intratumoral injection of CM. Results: In response to IR, NSCLC cell lines showed decreased SP-B regulated by the TGF-β signaling and decreased SP-B stimulated cell survival and epithelial-mesenchymal transition. Treatment with CM from irradiated cells activated sPLA2, enhanced protein kinase Cδ-MAPKs signaling pathway, and increased arachidonic acid production. We confirmed the in vivo roles of SP-B through mouse xenograft model. Conclusion: Our results revealed that down-regulation of SP-B was involved in the radiation-induced metastatic conversion of NSCLC and provided evidence that SP-B acted as a suppressor of NSCLC progression.

Title: A Mechanism Linking Extra Centrosomes to Chromosomal Instability Content: Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces three or more aneuploid daughter cells. Here we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. In contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly increased frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient 'multipolar spindle intermediate' in which merotelic kinetochore-microtubule attachment errors accumulate before centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours. We propose that this mechanism may be a common underlying cause of CIN in human cancer.

Title: Specific deficit of the ON response in visual transmission by targeted disruption of the mGIuR6 gene Content: Taking advantage of the restricted expression of metabotropic glutamate receptor subtype 6 (mGluR6) in retinal ON bipolar cells, we generated knockout mice lacking mGluR6 expression. The homozygous mutant mice showed a loss of ON responses but unchanged OFF responses to light. The mutant mice displayed no obvious changes in retinal cell organization nor in the projection of optic fibers to the brain. Furthermore, the mGluR6-deficient mice showed visual behavioral responses to light stimulation as examined by shuttle box avoidance behavior experiments using light exposure as a conditioned stimulus. The results demonstrate that mGluR6 is essential in synaptic transmission to the ON bipolar cell and that the OFF response provides an important means for transmitting visual information.

Title: In vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules Content: Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present several self- and foreign antigens to Kb-, Db-, or Qa-1b–restricted CD8+ cytotoxic T cells, presentation of some antigens was unaffected or significantly enhanced. Consistent with these findings, mice generated defective CD8+ T cell responses against class I–presented antigens. These findings reveal an important in vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.

Title: Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2. Content: Activating mutations within the K-ras gene occur in a high percentage of human pancreatic carcinomas. We reported previously that the presence of oncogenic, activated K-ras in human pancreatic carcinoma cell lines did not result in constitutive activation of the extracellular signal-regulated kinases (ERK1 and ERK2). In the present study, we further characterized the ERK signaling pathway in pancreatic tumor cell lines in order to determine whether the ERK pathway is subject to a compensatory downregulation. We found that the attenuation of serum-induced ERK activation was not due to a delay in the kinetics of ERK phosphorylation. Treatment with the tyrosine phosphatase inhibitor orthovanadate increased the level of ERK phosphorylation, implicating a vanadate-sensitive tyrosine phosphatase in the negative regulation of ERK. Furthermore, expression of a dual specificity phosphatase capable of inactivating ERK known as mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2) was elevated in most of the pancreatic tumor cell lines and correlated with the presence of active MAP kinase kinase (MEK). Taken together, these results suggest that pancreatic tumor cells expressing oncogenic K-ras compensate, in part, by upregulating the expression of MKP-2 to repress the ERK signaling pathway.

Title: MYC Disrupts the Circadian Clock and Metabolism in Cancer Cells. Content: The MYC oncogene encodes MYC, a transcription factor that binds the genome through sites termed E-boxes (5'-CACGTG-3'), which are identical to the binding sites of the heterodimeric CLOCK-BMAL1 master circadian transcription factor. Hence, we hypothesized that ectopic MYC expression perturbs the clock by deregulating E-box-driven components of the circadian network in cancer cells. We report here that deregulated expression of MYC or N-MYC disrupts the molecular clock in vitro by directly inducing REV-ERBα to dampen expression and oscillation of BMAL1, and this could be rescued by knockdown of REV-ERB. REV-ERBα expression predicts poor clinical outcome for N-MYC-driven human neuroblastomas that have diminished BMAL1 expression, and re-expression of ectopic BMAL1 in neuroblastoma cell lines suppresses their clonogenicity. Further, ectopic MYC profoundly alters oscillation of glucose metabolism and perturbs glutaminolysis. Our results demonstrate an unsuspected link between oncogenic transformation and circadian and metabolic dysrhythmia, which we surmise to be advantageous for cancer.

Title: A 40-Hz auditory potential recorded from the human scalp. Content: Computer techniques readily extract from the brainwaves an orderly sequence of brain potentials locked in time to sound stimuli. The potentials that appear 8 to 80 msec after the stimulus resemble 3 or 4 cycles of a 40-Hz sine wave; we show here that these waves combined to form a single, stable, composite wave when the sounds are repeated at rates around 40 per sec. This phenomenon, the 40-Hz event-related potential (ERP), displays several properties of theoretical and practical interest. First, it reportedly disappears with surgical anesthesia, and it resembles similar phenomena in the visual and olfactory system, facts which suggest that adequate processing of sensory information may require cyclical brain events in the 30- to 50-Hz range. Second, latency and amplitude measurements on the 40-Hz ERP indicate it may contain useful information on the number and basilar membrane location of the auditory nerve fibers a given tone excites. Third, the response is present at sound intensities very close to normal adult thresholds for the audiometric frequencies, a fact that could have application in clinical hearing testing.

Title: Light Activation of Channelrhodopsin-2 in Excitable Cells of Caenorhabditis elegans Triggers Rapid Behavioral Responses Content: For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.

Title: eRNAs are required for p53-dependent enhancer activity and gene transcription. Content: Binding within or nearby target genes involved in cell proliferation and survival enables the p53 tumor suppressor gene to regulate their transcription and cell-cycle progression. Using genome-wide chromatin-binding profiles, we describe binding of p53 also to regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound enhancer regions (p53BERs) indeed contain enhancer activity and interact intrachromosomally with multiple neighboring genes to convey long-distance p53-dependent transcription regulation. Furthermore, p53BERs produce, in a p53-dependent manner, enhancer RNAs (eRNAs) that are required for efficient transcriptional enhancement of interacting target genes and induction of a p53-dependent cell-cycle arrest. Thus, our results ascribe transcription enhancement activity to p53 with the capacity to regulate multiple genes from a single genomic binding site. Moreover, eRNA production from p53BERs is required for efficient p53 transcription enhancement.

Title: Extrathymic Generation of Regulatory T Cells in Placental Mammals Mitigates Maternal-Fetal Conflict Content: Regulatory T (Treg) cells, whose differentiation and function are controlled by X chromosome-encoded transcription factor Foxp3, are generated in the thymus (tTreg) and extrathymically (peripheral, pTreg), and their deficiency results in fatal autoimmunity. Here, we demonstrate that a Foxp3 enhancer, conserved noncoding sequence 1 (CNS1), essential for pTreg but dispensable for tTreg cell generation, is present only in placental mammals. CNS1 is largely composed of mammalian-wide interspersed repeats (MIR) that have undergone retrotransposition during early mammalian radiation. During pregnancy, pTreg cells specific to a model paternal alloantigen were generated in a CNS1-dependent manner and accumulated in the placenta. Furthermore, when mated with allogeneic, but not syngeneic, males, CNS1-deficient females showed increased fetal resorption accompanied by increased immune cell infiltration and defective remodeling of spiral arteries. Our results suggest that, during evolution, a CNS1-dependent mechanism of extrathymic differentiation of Treg cells emerged in placental animals to enforce maternal-fetal tolerance.

Title: ErbB4-neuregulin signaling modulates synapse development and dendritic arborization through distinct mechanisms. Content: 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.

Title: Differential effects of charybdotoxin on the activity of retinal ganglion cells in the dark- and light-adapted mouse retina Content: Patch-clamp recordings were made from retinal ganglion cells in the mouse retina. Under dark adaptation, blockage of BK(Ca) channels increases the spontaneous excitatory postsynaptic currents (EPSCs) and light-evoked On-EPSCs, while it decreases the light-evoked Off inhibitory postsynaptic currents (IPSCs). However, under light adaptation it decreases the light-evoked On-EPSCs, the spontaneous IPSCs and the light-evoked On- and Off-IPSCs. Blockage of BK(Ca) channels significantly altered the outputs of RGCs by changing their light-evoked responses into a bursting pattern and increasing the light-evoked depolarization of the membrane potentials, while it did not significantly change the peak firing rates of light-evoked responses.

Title: Embryonic Origin of Postnatal Neural Stem Cells Content: Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

Title: Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Content: Humoral immunity depends on both rapid and long-term antibody production against invading pathogens. This is achieved by the generation of spatially distinct extrafollicular plasmablast and follicular germinal center (GC) B cell populations, but the signals that guide responding B cells to these alternative compartments have not been fully elucidated. Here, we show that expression of the orphan G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) by activated B cells was essential for their movement to extrafollicular sites and induction of early plasmablast responses. Conversely, downregulation of EBI2 enabled B cells to access the center of follicles and promoted efficient GC formation. EBI2 therefore provides a previously uncharacterized dimension to B cell migration that is crucial for coordinating rapid versus long-term antibody responses.

Title: Substrate requirements for ErmC' methyltransferase activity. Content: ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.

Title: KAP1 controls endogenous retroviruses in embryonic stem cells Content: More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5′ untranslated region (5′UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5′UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

Title: ERAAP synergizes with MHC class I molecules to make the final cut in the antigenic peptide precursors in the endoplasmic reticulum. Content: The major histocompatibility complex class I molecules display peptides (pMHC I) on the cell surface for immune surveillance by CD8(+) T cells. These peptides are generated by proteolysis of intracellular polypeptides by the proteasome in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with antigen processing (ERAAP). To define the unknown mechanism of ERAAP function in vivo, we analyzed naturally processed peptides in cells with or without appropriate MHC I and ERAAP. In the absence of MHC I, ERAAP degraded the antigenic precursors in the ER. However, MHC I molecules could bind proteolytic intermediates and were essential for generation of the final peptide by ERAAP. Thus, ERAAP synergizes with MHC I to generate the final pMHC I repertoire.

Title: The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA Content: The product of the gene mutated in Bloom's syndrome, BLM, is a 3′–5′ DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg2+, is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.

Title: Continuous single-cell imaging of blood generation from haemogenic endothelium Content: Despite decades of research, the identity of the cells generating the first haematopoietic cells in mammalian embryos is unknown. Indeed, whether blood cells arise from mesodermal cells, mesenchymal progenitors, bipotent endothelial–haematopoietic precursors or haemogenic endothelial cells remains controversial. Proximity of endothelial and blood cells at sites of embryonic haematopoiesis, as well as their similar gene expression, led to the hypothesis of the endothelium generating blood. However, owing to lacking technology it has been impossible to observe blood cell emergence continuously at the single-cell level, and the postulated existence of haemogenic endothelial cells remains disputed. Here, using new imaging and cell-tracking methods, we show that embryonic endothelial cells can be haemogenic. By continuous long-term single-cell observation of mouse mesodermal cells generating endothelial cell and blood colonies, it was possible to detect haemogenic endothelial cells giving rise to blood cells. Living endothelial and haematopoietic cells were identified by simultaneous detection of morphology and multiple molecular and functional markers. Detachment of nascent blood cells from endothelium is not directly linked to asymmetric cell division, and haemogenic endothelial cells are specified from cells already expressing endothelial markers. These results improve our understanding of the developmental origin of mammalian blood and the potential generation of haematopoietic stem cells from embryonic stem cells.

Title: Abi1 is essential for the formation and activation of a WAVE2 signalling complex Content: WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2–Abi1–Nap1–PIR121 complex. The WAVE2–Abi1–Nap1–PIR121 complex is as active as the WAVE2–Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.

Title: Brief Communication Repeated Cocaine Administration Increases Voltage-Sensitive Calcium Currents in Response to Membrane Depolarization in Medial Prefrontal Cortex Pyramidal Neurons Content: The medial prefrontal cortex (mPFC) plays a critical role in cocaine addiction. However, evidence to elucidate how the mPFC is functionally involved in cocaine addiction remains incomplete. Recent studies have revealed that repeated cocaine administration induces various neuroadaptations in pyramidal mPFC neurons, including a reduction in voltage-gated K+ currents (VGKCs) and a possible increase in voltage-sensitive Ca2+ currents (I(Ca)). Here, we performed both current-clamp recordings in brain slices and voltage-clamp recordings in freshly dissociated cells to determine whether I(Ca) is altered in mPFC pyramidal neurons after chronic cocaine treatment with a short-term or long-term withdrawal. In addition, a critical role of VGKCs in regulating the generation of Ca2+ plateau potential was also studied in mPFC neurons. Repeated cocaine administration significantly prolonged the duration of evoked Ca2+ plateau potentials and increased the whole-cell I(Ca) in mPFC neurons after a 3 d withdrawal. Selective blockade of L-type Ca2+ channels by nifedipine not only significantly increased the threshold but also reduced the duration and amplitude of Ca2+ plateau potentials in both saline- and cocaine-withdrawn mPFC neurons. However, there was no significant difference in the increased threshold, reduced duration, and decreased amplitude of Ca2+ potentials between saline- and cocaine-withdrawn neurons after blockade of L-type Ca2+ channels. Moreover, an increase in amplitude was also observed, whereas the prolonged duration persisted, in Ca2+ potentials after 2-3 weeks of withdrawal. These findings indicate that chronic exposure to cocaine facilitates the responsiveness of I(Ca), particularly via the activated L-type Ca2+ channels, to excitatory stimuli in rat mPFC pyramidal neurons.

Title: Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation. Content: Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.

Title: High-Threshold Mechanosensitive Ion Channels Blocked by a Novel Conopeptide Mediate Pressure-Evoked Pain Content: Little is known about the molecular basis of somatosensory mechanotransduction in mammals. We screened a library of peptide toxins for effects on mechanically activated currents in cultured dorsal root ganglion neurons. One conopeptide analogue, termed NMB-1 for noxious mechanosensation blocker 1, selectively inhibits (IC50 1 µM) sustained mechanically activated currents in a subset of sensory neurons. Biotinylated NMB-1 retains activity and binds selectively to peripherin-positive nociceptive sensory neurons. The selectivity of NMB-1 was confirmed by the fact that it has no inhibitory effects on voltage-gated sodium and calcium channels, or ligand-gated channels such as acid-sensing ion channels or TRPA1 channels. Conversely, the tarantula toxin, GsMTx-4, which inhibits stretch-activated ion channels, had no effects on mechanically activated currents in sensory neurons. In behavioral assays, NMB-1 inhibits responses only to high intensity, painful mechanical stimulation and has no effects on low intensity mechanical stimulation or thermosensation. Unexpectedly, NMB-1 was found to also be an inhibitor of rapid FM1-43 loading (a measure of mechanotransduction) in cochlear hair cells. These data demonstrate that pharmacologically distinct channels respond to distinct types of mechanical stimuli and suggest that mechanically activated sustained currents underlie noxious mechanosensation. NMB-1 thus provides a novel diagnostic tool for the molecular definition of channels involved in hearing and pressure-evoked pain.

Title: Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Content: The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

Title: brain organization Content: Transient activation of estrogen receptors (ER) in the developing brain during a limited perinatal "window of time" is recognized as a key mechanism of defeminization of neural control of reproductive function and sexual behavior. Two major ER isoforms, alpha and beta, are present in neural circuits that govern ovarian cycle and sexual behavior. Using highly selective ER agonists, this study provides the first evidence for distinct contribution of individual ER isoforms to the process of estrogen dependent defeminization. Neonatal activation of the ERalpha in female rats resulted in abrogation of cyclic ovarian activity and female sexual behavior in adulthood. These effects are associated with male-like alterations in the morphology of the anteroventral periventricular (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA), as well as refractoriness to estrogen-mediated induction of sexual receptivity. Exposure to an ERbeta-selective agonist induced persistent estrus and had a strong defeminizing effect on the hypothalamic gonadotropin "surge generator" AVPV. However, neonatal ERbeta activation failed to alter female sexual behavior, responsiveness to estrogens and morphometric features of the behaviorally relevant SDN-POA. Thus, although co-present in several brain regions involved in the control of female reproductive function, ER isoforms convey different, and probably not synergistic, chemical signals in the course of neonatal sex-specific brain organization.

Title: Latent membrane protein 1 of Epstein–Barr virus coordinately regulates proliferation with control of apoptosis Content: Latent membrane protein 1 (LMP1), an oncoprotein encoded by Epstein–Barr virus (EBV), is an integral membrane protein, which acts like a constitutively active receptor. LMP1 is critical for some facet of EBV's induction and maintenance of proliferation of infected B cells. It, in part, mimics signaling by the CD40 receptor and has been implicated in regulating proliferation, survival, or both properties of EBV-infected cells. We established a conditional LMP1 allele in the context of the intact EBV genome to define the immediate-early cellular target genes regulated by LMP1 in order to assess its contributions to infected human B cells. The functional analysis of this conditional system indicated that LMP1 specifically induces mitogenic B-cell activation through c-myc and Jun/AP1 family members and confirms its direct role in upregulating expression of multiple genes with opposing activities involved in cell survival. LMP1's signals were found to be essential for the G1/S transition in human B cells; cells lacking LMP1's signals are cell cycle arrested and survive quiescently. LMP1's activities are therefore not required to maintain survival in nonproliferating cells. LMP1 does induce both pro- and antiapoptotic genes whose balance seems to permit survival during LMP1's induction and maintenance of proliferation.

Title: Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling Content: Schizophrenia is a complex disorder that interferes with the function of several brain systems required for cognition and normal social behaviour. Although the most notable clinical aspects of the disease only become apparent during late adolescence or early adulthood, many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder with a strong genetic component. Several independent studies have identified neuregulin 1 (NRG1) and its receptor ERBB4 as important risk genes for schizophrenia, although their precise role in the disease process remains unknown. Here we show that Nrg1 and ErbB4 signalling controls the development of inhibitory circuitries in the mammalian cerebral cortex by cell-autonomously regulating the connectivity of specific GABA (γ-aminobutyric acid)-containing interneurons. In contrast to the prevalent view, which supports a role for these genes in the formation and function of excitatory synapses between pyramidal cells, we found that ErbB4 expression in the mouse neocortex and hippocampus is largely confined to certain classes of interneurons. In particular, ErbB4 is expressed by many parvalbumin-expressing chandelier and basket cells, where it localizes to axon terminals and postsynaptic densities receiving glutamatergic input. Gain- and loss-of-function experiments, both in vitro and in vivo, demonstrate that ErbB4 cell-autonomously promotes the formation of axo-axonic inhibitory synapses over pyramidal cells, and that this function is probably mediated by Nrg1. In addition, ErbB4 expression in GABA-containing interneurons regulates the formation of excitatory synapses onto the dendrites of these cells. By contrast, ErbB4 is dispensable for excitatory transmission between pyramidal neurons. Altogether, our results indicate that Nrg1 and ErbB4 signalling is required for the wiring of GABA-mediated circuits in the postnatal cortex, providing a new perspective to the involvement of these genes in the aetiology of schizophrenia.

Title: BRAF mutation predicts for MEK-dependence in non-small cell lung cancer (NSCLC). Content: 5247 Constitutive ERK signaling is common in human cancer and is often the result of activating mutations of BRAF, RAS and upstream receptor tyrosine kinases. Missense BRAF kinase domain mutations are frequently observed in melanoma, colon and thyroid cancers and less frequently in lung and other cancer types. The vast majority (>90%) involve a glutamic acid for valine substitution at codon 600 (V600E), which results in elevated BRAF kinase activity. BRAF kinase domain mutations with intermediate and impaired kinase activity have also been identified, most frequently in NSCLC. We have previously reported that tumors with V600E BRAF mutation are selectively sensitive to MEK inhibition. Using the potent and selective MEK1/2 inhibitor PD0325901 (Pfizer), we examined a panel of NSCLC cell lines with mutant EGFR, KRAS, and/or low, intermediate and high-activity BRAF kinase domain mutations for MEK dependence. In all but one case, EGFR, KRAS and BRAF mutations were mutually exclusive with the exception being a cell line with concurrent NRAS and intermediate activity BRAF mutations. Consistent with our prior results, NSCLC cells with V600E BRAF mutation were exquisitely sensitive to MEK inhibition (PD0325901 IC50 of 2nM). The proliferation of cells with non-V600E mutations, including those with high (G469A), intermediate (L597V) and impaired (G466V) kinase activities, was also MEK dependent with IC50’s ranging between 2.7 and 80 nM. Inhibition of MEK in these cells resulted in downregulation of cyclin D1 and G1 growth arrest, with variable induction of apoptosis. Despite high basal ERK activity, NSCLC tumor cells with EGFR mutation were uniformly resistant to MEK inhibition (at doses of up to 500nM), despite effective and prolonged inhibition of ERK phosphorylation. Tumor cells with RAS mutation had a more variable response, with some cell lines demonstrating sensitivity, while others were completely resistant. There was no correlation between basal ERK activity and sensitivity to MEK inhibition. A strong inverse correlation between Akt activity and PD0325901 sensitivity was observed. These results suggest that MEK inhibition may be useful therapeutically in tumors with V600E and non-V600E BRAF kinase domain mutations. The results also suggest that inhibition of both MEK and Akt signaling may be required in NSCLC tumors with high basal AKT activity.

Title: Vision tests in the mouse: Functional phenotyping with electroretinography. Content: Electroretinography (ERG) is an established diagnostic technique in clinical ophthalmology and provides objective information about retinal function. This technique is also applied in basic research, where animal models of hereditary retinopathies have significantly contributed to our understanding of the composition of ERG responses in general and how retinal degenerative pathologies alter retinal function specifically. Indeed, electrophysiologic assessment of transgenic mice, which are genetically engineered to mimic human mutations that lead to retinal diseases, can be well compared with clinical data. Furthermore, limitations on examinations (e.g. length of measurement, range of light intensity) are much less of a concern when assessing mice compared to human patients. In order to measure and analyze retinal responses properly, several important aspects have to be considered. This paper focuses on these aspects, and shows exemplary ERG data which were obtained from normal wild-type mice and from transgenic mice with specific functional properties, namely Rho-/- (rod opsin knockout, cone function only), and Cnga3-/- (cone CNG channel deficient, rod function only) to illustrate rod and cone system contributions to ERG responses.

Title: 7α, 25-dihydroxycholesterol-mediated activation of EBI2 in immune regulation and diseases Content: EBI2, aka GPR183, is a G-couple receptor originally identified in 1993 as one of main genes induced in Burkitt's lymphoma cell line BL41 by Epstein-Barr virus (EBV) infection. After it was reported in 2009 that the receptor played a key role in regulating B cell migration and responses, we initiated an effort in looking for its endogenous ligand. In 2011 we and another group reported the identification of 7α, 25-dihydroxyxcholesterol (7α, 25-OHC), an oxysterol, as the likely physiological ligand of EBI2. A few subsequently published studies further elucidated how 7α, 25-OHC bound to EBI2, and how a gradient of 7α, 25-OHC could be generated in vivo and regulated migration, activation, and functions of B cells, T cells, dendritic cells (DCs), monocytes/macrophages, and astrocytes. The identification of 7α, 25-OHC as a G protein-coupled receptor ligand revealed a previously unknown signaling system of oxysterols, a class of molecules which exert profound biological functions. Dysregulation of the synthesis or functions of these molecules is believed to contribute to inflammation and autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, cancer as well as metabolic diseases such as diabetes, obesity, and dyslipidemia. Therefore EBI2 may represent a promising target for therapeutic interventions for human diseases.

Title: Converging Intracranial Markers of Conscious Access Content: We compared conscious and nonconscious processing of briefly flashed words using a visual masking procedure while recording intracranial electroencephalogram (iEEG) in ten patients. Nonconscious processing of masked words was observed in multiple cortical areas, mostly within an early time window (<300 ms), accompanied by induced gamma-band activity, but without coherent long-distance neural activity, suggesting a quickly dissipating feedforward wave. In contrast, conscious processing of unmasked words was characterized by the convergence of four distinct neurophysiological markers: sustained voltage changes, particularly in prefrontal cortex, large increases in spectral power in the gamma band, increases in long-distance phase synchrony in the beta range, and increases in long-range Granger causality. We argue that all of those measures provide distinct windows into the same distributed state of conscious processing. These results have a direct impact on current theoretical discussions concerning the neural correlates of conscious access.

Title: BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells into Embryonic and Extraembryonic Lineages Content: BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.

Title: BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax Content: BCL-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma1–3. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths4–13. An emerging family of Bcl-2 -related proteins share two highly conserved regions14–20 referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-214. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitu-tion of Gly 145 in BHl domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in inter-leukin-3 deprivation, γ-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homo-dimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.

Title: Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Content: Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.

Title: OVEREXPRESSION OF BOTH CLOCK AND BMAL1 INHIBITS ENTRY TO S PHASE IN HUMAN COLON CANCER CELLS. Content: Many physiological, biochemical and behavioral processes operate under the circadian rhythm, which is generated by an internal time-keeping mechanism commonly referred to as the biological clock, in almost all organisms from bacteria to mammals. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop, in which CLOCK and BMAL1 are positive regulators. A cell has two mechanisms, "cell cycle" and "cell rhythm", the relationship between which remains controversial. Therefore, the aim of this study was to explore the effect of Clock and Bmal1 on cell cycle, especially on the G1 phase, using vectors with the tetracycline operator-repressor system. The present study revealed that simultaneous induction of Bmal1 and Clock had an influential effect on the cell cycle in SW480/T-REx/Clock/Bmal1 cells, in which both Clock and Bmal1 could be induced by tetracycline. The observation that induction of both Clock and Bmal1 inhibited cell growth and the significant increase of the G1 phase proportion of in SW480/T-REx/Clock/Bmal1 cells indicated that entry from the G1 to S phase was inhibited by the induction of Clock and Bmal1. Furthermore, overexpression of Clock and Bmal1 prevented the cells from entering into the G2/M phase induced by Paclitaxel, and made the cells more resistant to the agent. In conclusion, we found that overexpression of both Clock and Bmal1 suppressed cell growth. In addition, the present study raised the possibility that Clock and Bmal1 may in part play a role in preventing the cells from entering G1 to S phase of cell cycle via suppression of CyclinD1 expression, and thus acquiring resistance to Paclitaxel.

Title: Peripheral B cell subsets. Content: Our understanding of the origins and the biological functions of different peripheral B cell subsets continues to evolve. Some understanding has been obtained regarding the synergy between BCR-derived signals and other receptors and signaling pathways that drive the development of follicular, marginal zone, and B-1 B cells, but this remains a complex and poorly understood issue. More recent information regarding the origins of B-1 and B-2 B cells, the ability of follicular B cells to mature both in the bone marrow and the spleen, the existence of a definable precursor for MZ B cells, and the ability of follicular B cells to occupy two distinct niches are all highlighted in this review.

Title: B-cell self-tolerance in humans. Content: Two mechanisms account for generation of the human antibody repertoire; V(D)J recombination during the early stages of B-cell development in the bone marrow and somatic mutation of immunoglobulin genes in mature B cells responding to antigen in the periphery. V(D)J recombination produces diversity by random joining of gene segments and somatic mutation by introducing random point mutations. Both are required to attain the degree of antigen receptor diversification that is necessary for immune protection: defects in either mechanism are associated with increased susceptibility to infection. However, the downside of producing enormous random diversity in the antibody repertoire is the generation of autoantibodies. To prevent autoimmunity B cells expressing autoantibodies are regulated by strict mechanisms that either modify the specificity of autoantibodies or the fate of cells expressing such antibodies. Abnormalities in B-cell self-tolerance are associated with a large number of autoimmune diseases, but the precise nature of the defects is less well defined. Here we summarize recent data on the self-reactive B-cell repertoire in healthy humans and in patients with autoimmunity.

Title: Quiescent and active hippocampal neural stem cells with distinct morphologies respond selectively to physiological and pathological stimuli and aging. Content: New neurons are generated in the adult hippocampus throughout life by neural stem/progenitor cells (NSCs), and neurogenesis is a plastic process responsive to external stimuli. We show that canonical Notch signaling through RBP-J is required for hippocampal neurogenesis. Notch signaling distinguishes morphologically distinct Sox2(+) NSCs, and within these pools subpopulations can shuttle between mitotically active or quiescent. Radial and horizontal NSCs respond selectively to neurogenic stimuli. Physical exercise activates the quiescent radial population whereas epileptic seizures induce expansion of the horizontal NSC pool. Surprisingly, reduced neurogenesis correlates with a loss of active horizontal NSCs in aged mice rather than a total loss of stem cells, and the transition to a quiescent state is reversible to rejuvenate neurogenesis in the brain. The discovery of multiple NSC populations with Notch dependence but selective responses to stimuli and reversible quiescence has important implications for the mechanisms of adaptive learning and also for regenerative therapy.

Title: Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons Content: During corticogenesis, pyramidal neurons (∼80% of cortical neurons) arise from the ventricular zone, pass through a multipolar stage to become bipolar and attach to radial glia, and then migrate to their proper position within the cortex. As pyramidal neurons migrate radially, they remain attached to their glial substrate as they pass through the subventricular and intermediate zones, regions rich in tangentially migrating interneurons and axon fibre tracts. We examined the role of lamellipodin (Lpd), a homologue of a key regulator of neuronal migration and polarization in Caenorhabditis elegans, in corticogenesis. Lpd depletion caused bipolar pyramidal neurons to adopt a tangential, rather than radial-glial, migration mode without affecting cell fate. Mechanistically, Lpd depletion reduced the activity of SRF, a transcription factor regulated by changes in the ratio of polymerized to unpolymerized actin. Therefore, Lpd depletion exposes a role for SRF in directing pyramidal neurons to select a radial migration pathway along glia rather than a tangential migration mode.

Title: Therapeutic potential of mood stabilizers lithium and valproic acid: beyond bipolar disorder. Content: The mood stabilizers lithium and valproic acid (VPA) are traditionally used to treat bipolar disorder (BD), a severe mental illness arising from complex interactions between genes and environment that drive deficits in cellular plasticity and resiliency. The therapeutic potential of these drugs in other central nervous system diseases is also gaining support. This article reviews the various mechanisms of action of lithium and VPA gleaned from cellular and animal models of neurologic, neurodegenerative, and neuropsychiatric disorders. Clinical evidence is included when available to provide a comprehensive perspective of the field and to acknowledge some of the limitations of these treatments. First, the review describes how action at these drugs' primary targets--glycogen synthase kinase-3 for lithium and histone deacetylases for VPA--induces the transcription and expression of neurotrophic, angiogenic, and neuroprotective proteins. Cell survival signaling cascades, oxidative stress pathways, and protein quality control mechanisms may further underlie lithium and VPA's beneficial actions. The ability of cotreatment to augment neuroprotection and enhance stem cell homing and migration is also discussed, as are microRNAs as new therapeutic targets. Finally, preclinical findings have shown that the neuroprotective benefits of these agents facilitate anti-inflammation, angiogenesis, neurogenesis, blood-brain barrier integrity, and disease-specific neuroprotection. These mechanisms can be compared with dysregulated disease mechanisms to suggest core cellular and molecular disturbances identifiable by specific risk biomarkers. Future clinical endeavors are warranted to determine the therapeutic potential of lithium and VPA across the spectrum of central nervous system diseases, with particular emphasis on a personalized medicine approach toward treating these disorders.

Title: The cell cycle in polyploid megakaryocytes is associated with reduced activity of cyclin B1-dependent cdc2 kinase. Content: The platelet precursor, the megakaryocyte, matures to a polyploid cell as a result of DNA replication in the absence of mitosis (endomitosis). The factors controlling endomitosis are accessible to analysis in our megakaryocytic cell line, MegT, generated by targeted expression of temperature-sensitive simian virus 40 large T antigen to megakaryocytes of transgenic mice. We aimed to define whether endomitosis consists of a continuous phase of DNA synthesis (S) or of S phases interrupted by gaps. Analysis of the cell cycle in MegT cells revealed that, upon inactivation of large T antigen, the cells shifted from a mitotic cell cycle to an endomitotic cell cycle consisting of S/Gap phases. The level of the G1/S cyclin, cyclin A, as well as of the G1 phase cyclin, cyclin D3, were elevated at the onset of DNA synthesis, either in MegT cells undergoing a mitotic cell cycle or during endomitosis. In contrast, the level of the mitotic cyclin, cyclin B1, cycled in cells displaying a mitotic cell cycle while not detectable during endomitosis. Comparable levels of the mitotic kinase protein, Cdc2, were detected during the mitotic cell cycle or during endomitosis; however, cyclin B1-dependent Cdc2 kinase activity was largely abolished in the polyploid cells. Fibroblasts immortalized with the same heat-labile oncogene do not display reduced levels of cyclin B1 upon shifting to high temperature nor do they become polyploid, indicating that reduced levels of cyclin B1 is a property of megakaryocytes and not of the T-antigen mutant. We conclude that cellular programming during endoreduplication in megakaryocytes is associated with reduced levels of cyclin B1.

Title: Ciliary Extracellular Vesicles: Txt Msg Organelles Content: Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV–cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia–EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies.

Title: Discrimination of speech stimuli based on neuronal response phase patterns depends on acoustics but not comprehension. Content: Speech stimuli give rise to neural activity in the listener that can be observed as waveforms using magnetoencephalography. Although waveforms vary greatly from trial to trial due to activity unrelated to the stimulus, it has been demonstrated that spoken sentences can be discriminated based on theta-band (3-7 Hz) phase patterns in single-trial response waveforms. Furthermore, manipulations of the speech signal envelope and fine structure that reduced intelligibility were found to produce correlated reductions in discrimination performance, suggesting a relationship between theta-band phase patterns and speech comprehension. This study investigates the nature of this relationship, hypothesizing that theta-band phase patterns primarily reflect cortical processing of low-frequency (<40 Hz) modulations present in the acoustic signal and required for intelligibility, rather than processing exclusively related to comprehension (e.g., lexical, syntactic, semantic). Using stimuli that are quite similar to normal spoken sentences in terms of low-frequency modulation characteristics but are unintelligible (i.e., their time-inverted counterparts), we find that discrimination performance based on theta-band phase patterns is equal for both types of stimuli. Consistent with earlier findings, we also observe that whereas theta-band phase patterns differ across stimuli, power patterns do not. We use a simulation model of the single-trial response to spoken sentence stimuli to demonstrate that phase-locked responses to low-frequency modulations of the acoustic signal can account not only for the phase but also for the power results. The simulation offers insight into the interpretation of the empirical results with respect to phase-resetting and power-enhancement models of the evoked response.

Title: RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Content: Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5'-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.

Title: Infralimbic BDNF/TrkB enhancement of GluN2B currents facilitates extinction of a cocaine-conditioned place preference. Content: Brain-derived neurotrophic factor (BDNF) regulates synaptic activity and behavioral flexibility, and reduction of BDNF strongly predicts psychiatric disorders and cognitive dysfunction. Restoration of BDNF-dependent activity could alleviate these impairments, but BDNF has limited clinical utility due to its pharmacokinetics. Here we demonstrate that activation of a primary BDNF target, the tropomyosin-related kinase B (TrkB) receptor, enhances the amplitude and prolongs the decay kinetics of N-methyl-d-aspartate receptor (NMDAR) currents in male rat infralimbic prefrontal pyramidal neurons. Moreover, these effects were prevented and reversed by blockade of NMDARs containing the GluN2B subunit. Our results show that this signaling cascade bidirectionally regulates extinction of a cocaine-induced conditioned place preference (CPP), a task that requires behavioral flexibility. Blockade of infralimbic TrkB receptors or GluN2B-containing NMDARs disrupted consolidation of extinction of the CPP. In contrast, extinction was strengthened by potentiation of TrkB receptor activity with infralimbic infusions of BDNF or systemic injections of 7,8 dihydroxyflavone (7,8DHF), the newly synthesized TrkB receptor agonist. The 7,8DHF-induced enhancement of extinction was prevented by infralimbic infusions of a GluN2B-specific receptor antagonist, demonstrating that TrkB receptor activation enhances extinction of cocaine-CPP via GluN2B-containing NMDARs. Together, infralimbic TrkB receptor activation strengthens GluN2B-containing NMDAR currents to support extinction learning. TrkB receptor agonists would therefore be useful as pharmacological adjuncts for extinction-based therapies for treatment of psychiatric disorders associated with reduced BDNF activity.

Title: Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks. Content: The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.

Title: Distinct memory CD4+ T cells with commitment to T follicular helper- and T helper 1-cell lineages are generated after acute viral infection. Content: CD4(+) T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for long-lived antibody responses. However, it remains unclear whether there are CD4(+) memory T cells committed to the Tfh cell lineage after antigen clearance. By using adoptive transfer of antigen-specific memory CD4(+) T cell subpopulations in the lymphocytic choriomeningitis virus infection model, we found that there are distinct memory CD4(+) T cell populations with commitment to either Tfh- or Th1-cell lineages. Our conclusions are based on gene expression profiles, epigenetic studies, and phenotypic and functional analyses. Our findings indicate that CD4(+) memory T cells "remember" their previous effector lineage after antigen clearance, being poised to reacquire their lineage-specific effector functions upon antigen reencounter. These findings have important implications for rational vaccine design, where improving the generation and engagement of memory Tfh cells could be used to enhance vaccine-induced protective immunity.

Title: Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism. Content: BACKGROUND & AIMS GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

Title: Finding the right niche: B-cell migration in the early phases of T-dependent antibody responses. Content: Humoral immune responses depend on B cells encountering antigen, interacting with helper T cells, proliferating and differentiating into low-affinity plasma cells or, after organizing into a germinal center (GC), high-affinity plasma cells and memory B cells. Remarkably, each of these events occurs in association with distinct stromal cells in separate subcompartments of the lymphoid tissue. B cells must migrate from niche to niche in a rapid and highly regulated manner to successfully mount a response. The chemokine, CXCL13, plays a central role in guiding B cells to follicles whereas T-zone chemokines guide activated B cells to the T zone. Sphingosine-1-phosphate (S1P) promotes cell egress from the tissue, as well as marginal-zone B-cell positioning in the spleen. Recent studies have identified a role for the orphan receptor, EBV-induced molecule 2 (EBI2; GPR183), in guiding activated B cells to inter and outer follicular niche(s) and down-regulation of this receptor is essential for organizing cells into GCs. In this review, we discuss current understanding of the roles played by chemokines, S1P and EBI2 in the migration events that underlie humoral immune responses.

Title: Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic Ras is a dominant predictor for resistance. Content: The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser(473)-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Ras-dependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.

Title: Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors Content: Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination.

Title: UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein. Content: We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.

Title: The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins Content: How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.

Title: Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells Content: Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

Title: CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling via transactivation of GSK-3β and Axin2 expression Content: Caudal-related homeobox transcription factor 2 (CDX2), an intestine-specific nuclear transcription factor, has been strongly implicated in the tumourigenesis of various human cancers. However, the functional role of CDX2 in the development and progression of colorectal cancer (CRC) is not well known. In this study, CDX2 knockdown in colon cancer cells promoted cell proliferation in vitro, accelerated tumor formation in vivo, and induced a cell cycle transition from G0/G1 to S phase, whereas CDX2 overexpression inhibited cell proliferation. TOP/FOP-Flash reporter assay showed that CDX2 knockdown or CDX2 overexpression significantly increased or decreased Wnt signaling activity. Western blot assay showed that downstream targets of Wnt signaling, including β-catenin, cyclin D1 and c-myc, were up-regulated or down-regulated in CDX2-knockdown or CDX2-overexpressing colon cancer cells. In addition, suppression of Wnt signaling by XAV-939 led to a marked suppression of the cell proliferation enhanced by CDX2 knockdown, whereas activation of this signaling by CHIR-99021 significantly enhanced the cell proliferation inhibited by CDX2 overexpression. Dual-luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays further confirmed that CDX2 transcriptionally activates glycogen synthase kinase-3β (GSK-3β) and axis inhibition protein 2 (Axin2) expression by directly binding to the promoter of GSK-3β and the upstream enhancer of Axin2. In conclusion, these results indicated that CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling.

Title: BLM helicase facilitates telomere replication during leading strand synthesis of telomeres Content: Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome-associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.

Title: Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells Content: The reorientation of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell enables the directional secretion of cytokines and lytic factors. By single-cell photoactivation of the T cell antigen receptor, we show that MTOC polarization is driven by localized accumulation of diacylglycerol (DAG). MTOC reorientation was closely preceded first by production of DAG and then by recruitment of the microtubule motor protein dynein. Blocking DAG production or disrupting the localization of DAG impaired MTOC recruitment. Localized DAG accumulation was also required for cytotoxic T cell–mediated killing. Furthermore, photoactivation of DAG itself was sufficient to induce transient polarization. Our data identify a DAG-dependent pathway that signals through dynein to control microtubule polarity in T cells.

Title: Microtubule sliding activity of a kinesin-8 promotes spindle assembly and spindle length control Content: Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide-disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

Title: Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal. Content: Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.

Title: Interneuronal DISC1 regulates NRG1-ErbB4 signalling and excitatory-inhibitory synapse formation in the mature cortex. Content: Neuregulin-1 (NRG1) and its receptor ErbB4 influence several processes of neurodevelopment, but the mechanisms regulating this signalling in the mature brain are not well known. DISC1 is a multifunctional scaffold protein that mediates many cellular processes. Here we present a functional relationship between DISC1 and NRG1-ErbB4 signalling in mature cortical interneurons. By cell type-specific gene modulation in vitro and in vivo including in a mutant DISC1 mouse model, we demonstrate that DISC1 inhibits NRG1-induced ErbB4 activation and signalling. This effect is likely mediated by competitive inhibition of binding of ErbB4 to PSD95. Finally, we show that interneuronal DISC1 affects NRG1-ErbB4-mediated phenotypes in the fast spiking interneuron-pyramidal neuron circuit. Post-mortem brain analyses and some genetic studies have reported interneuronal deficits and involvement of the DISC1, NRG1 and ErbB4 genes in schizophrenia, respectively. Our results suggest a mechanism by which cross-talk between DISC1 and NRG1-ErbB4 signalling may contribute to these deficits.

Title: Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction. Content: AIMS Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents. METHODS AND RESULTS CPCs were derived from atrial appendage explants from patients who underwent heart valve surgery. CPC-conditioned medium (CM) inhibited apoptosis in mouse HL-1 cardiomyocytic cells, while enhancing tube formation in human umbilical vein endothelial cells. These effects were abrogated by depleting CM of EVs. They were reproduced by EVs secreted by CPCs, but not by those secreted by human dermal fibroblasts. Transmission electron microscopy and nanoparticle tracking analysis showed most EVs to be 30-90 nm in diameter, the size of exosomes, although smaller and larger vesicles were also present. MicroRNAs most highly enriched in EVs secreted by CPCs compared with fibroblasts included miR-210, miR-132, and miR-146a-3p. miR-210 down-regulated its known targets, ephrin A3 and PTP1b, inhibiting apoptosis in cardiomyocytic cells. miR-132 down-regulated its target, RasGAP-p120, enhancing tube formation in endothelial cells. Infarcted hearts injected with EVs from CPCs, but not from fibroblasts, exhibited less cardiomyocyte apoptosis, enhanced angiogenesis, and improved LV ejection fraction (0.8 ± 6.8 vs. -21.3 ± 4.5%; P < 0.05) compared with those injected with control medium. CONCLUSION EVs are the active component of the paracrine secretion by human CPCs. As a cell-free approach, EVs could circumvent many of the limitations of cell transplantation.

Title: EB virus-encoded RNAs are recognized by RIG-I and activate signaling to induce type I IFN. Content: Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV-infected cells, and are expected to show secondary structures with many short stem-loops. Retinoic acid-inducible gene I (RIG-I) is a cytosolic protein that detects viral double-stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG-I as dsRNA. Transfection of RIG-I plasmid induced IFNs and IFN-stimulated genes (ISGs) in EBV-positive Burkitt's lymphoma (BL) cells, but not in their EBV-negative counterparts or EBER-knockout EBV-infected BL cells. Transfection of EBER plasmid or in vitro-synthesized EBERs induced expression of type I IFNs and ISGs in RIG-I-expressing, EBV-negative BL cells, but not in RIG-I-minus counterparts. EBERs activated RIG-I's substrates, NF-kappaB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co-precipitated with RIG-I. These results indicate that EBERs are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected cells.

Title: Alterations on peripheral B cell subsets following an acute uncomplicated clinical malaria infection in children Content: BACKGROUND The effects of Plasmodium falciparum on B-cell homeostasis have not been well characterized. This study investigated whether an episode of acute malaria in young children results in changes in the peripheral B cell phenotype. METHODS Using flow-cytofluorimetric analysis, the B cell phenotypes found in the peripheral blood of children aged 2-5 years were characterized during an episode of acute uncomplicated clinical malaria and four weeks post-recovery and in healthy age-matched controls. RESULTS There was a significant decrease in CD19+ B lymphocytes during acute malaria. Characterization of the CD19+ B cell subsets in the peripheral blood based on expression of IgD and CD38 revealed a significant decrease in the numbers of naive 1 CD38-IgD+ B cells while there was an increase in CD38+IgD- memory 3 B cells during acute malaria. Further analysis of the peripheral B cell phenotype also identified an expansion of transitional CD10+CD19+ B cells in children following an episode of acute malaria with up to 25% of total CD19+ B cell pool residing in this subset. CONCLUSION Children experiencing an episode of acute uncomplicated clinical malaria experienced profound disturbances in B cell homeostasis.

Title: The Spemann Organizer Signal noggin Binds and Inactivates Bone Morphogenetic Protein 4 Content: Signals released by the Spemann organizer of the amphibian gastrula can directly induce neural tissue from ectoderm and can dorsalize ventral mesoderm to form muscle. The secreted polypeptide noggin mimics these activities and is expressed at the appropriate time and place to participate in the organizer signal. Neural induction and mesoderm dorsalization are antagonized by bone morphogenetic proteins (BMPs), which induce epidermis and ventral mesoderm instead. Here we report that noggin protein binds BMP4 with high affinity and can abolish BMP4 activity by blocking binding to cognate cell-surface receptors. These data suggest that noggin secreted by the organizer patterns the embryo by interrupting BMP signaling.

Title: Integrins promote cytokinesis through the RSK signaling axis. Content: Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.

Title: SLP-65 regulates immunoglobulin light chain gene recombination through the PI(3)K-PKB-Foxo pathway Content: Although the essential role of the adaptor protein SLP-65 in pre-B cell differentiation is established, the molecular mechanism underlying its function is poorly understood. In this study, we uncover a link between SLP-65–dependent signaling and the phosphoinositide-3-OH kinase (PI(3)K)–protein kinase B (PKB)–Foxo pathway. We show that the forkhead box transcription factor Foxo3a promotes light chain rearrangement in pre-B cells. Our data suggest that PKB suppresses light chain recombination by phosphorylating Foxo proteins, whereas reconstitution of SLP-65 function counteracts PKB activation and promotes Foxo3a and Foxo1 activity in pre-B cells. Together, these data illuminate a molecular function of SLP-65 and identify a key role for Foxo proteins in the regulation of light chain recombination, receptor editing and B cell selection.

Title: Lung Carcinogenesis by Diesel Exhaust Particles and the Carcinogenic Mechanism Via Active Oxygens. Content: In an experiment to clarify the involvement of oxygen radicals in lung carcinogenesis induced by diesel exhaust particles (DEP), we found that there is a strong relation between lung tumor response and formation of 8-hydroxydeoxyguanosine (8-OHdG) in lung DNA of mice administered DEP by repeated intratracheal instillation. Repeated intratracheal instillation of DEP also induced the activity of cytochrome P-450 reductase in the lungs as a representative enzyme of superoxide generation, and two types of nitric oxide (NO) synthase, cNOS and iNOS, in the lungs. On the other hand, activities of CuZn-superoxide dismutase (SOD) and Mn-SOD antioxidant enzymes were depressed by the instillation of DEP. These results suggest that generation of superoxide, hydroxyI radical, and nitric oxide are increased in epithelial cells in airways, and that the increased superoxide and nitric oxide react very easily to produce peroxynitrite (ONOO(-)). The peroxynitrite also produce hydroxyI radical. The hydroxyl radical may play an important role in carcinogenesis by DEP.

Title: Whole-genome fingerprint of the DNA methylome during human B cell differentiation Content: We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, which was associated with upregulation of key B cell transcription factors and affected multiple genes involved in B cell biology. Late differentiation stages, in contrast, showed extensive demethylation of heterochromatin and methylation gain at Polycomb-repressed areas, and genes with apparent functional impact in B cells were not affected. This signature, which has previously been linked to aging and cancer, was particularly widespread in mature cells with an extended lifespan. Comparing B cell neoplasms with their normal counterparts, we determined that they frequently acquire methylation changes in regions already undergoing dynamic methylation during normal B cell differentiation.

Title: Discovery of endogenous catecholamines in lymphocytes and evidence for catecholamine regulation of lymphocyte function via an autocrine loop. Content: Evidence has been obtained that catecholamines and their metabolites are present in single lymphocytes and extracts of T- and B-cell clones by use of capillary electrophoresis with electrochemical detection. Pharmacological inhibition of tyrosine hydroxylase reduces observed catecholamine levels, suggesting catecholamine synthesis by lymphocytes. Intracellular dopamine levels are shown to be increased by extra-cellular dopamine, suggesting a cellular-uptake mechanism. Furthermore, incubation with either dopamine or L-dihydroxyphenylalanine, a precursor of dopamine, results in a dose-dependent inhibition of lymphocyte proliferation and differentiation. Together, these results suggest the presence of an autocrine loop whereby lymphocytes down-regulate their own activity.

Title: The modulation of human natural killer cell activity by prostaglandins. Content: Prostaglandins (PGs) have been implicated as a regulator of tumor growth in mice and humans. Since natural killer cell (NK) cytotoxicity may be an important component of immune surveillance against cancer, it is appropriate to study whether the amount of PGs produced by tumors may be sufficient to suppress NK activity. Accordingly, the effect of various PGs on the NK activity of human peripheral mononuclear cells was investigated. The percentage cytotoxicity was measured by the release of Cr51 from labeled K562 and other target cells. At very high concentrations of PG (10(-6) M), suppression was seen with PGE2, PGD2, PGA2, and PGF2 alpha. However, at concentrations of PG in the physiologic range (10(-8) M), significant suppression was seen with PGE2 and PGD2 only. The percentage suppression with PGE2 ranged from 77% to 9.5% over a range of concentrations from 10(-5) to 10(-9) M (45% at 10(-8) M). Significant suppression was observed at 10(-8) M PGE2 with 4 different targets and at effector:target ratios varying from 50:1 to 12.5:1. To assess whether the suppressive effect of PGE2 was directed at the effector and/or target cell, K562 cells or effector cells were pretreated with PGE2. Significant suppression was seen with effector cell pretreatment but not with target cell pretreatment. Finally, the suppressive effects of supernatants obtained from tumor cell lines (polyoma virus-transformed murine fibroblast cell line, PY3T3) was determined. The marked suppressive effect of the supernatant could be attributed to its content of PGE. Thus, it appears that the production of PGE by tumor cells may be an important modulator of human NK activity.

Title: Integrin activation by Fam38A uses a novel mechanism of R-Ras targeting to the endoplasmic reticulum. Content: The integrin family of heterodimeric cell-surface receptors are fundamental in cell-cell and cell-matrix adhesion. Changes to either integrin-ligand affinity or integrin gene expression are central to a variety of disease processes, including inflammation, cardiovascular disease and cancer. In screening for novel activators of integrin-ligand affinity we identified the previously uncharacterised multi-transmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER). siRNA knockdown of Fam38A in epithelial cells inactivates endogenous beta1 integrin, reducing cell adhesion. Fam38A mediates integrin activation by recruiting the small GTPase R-Ras to the ER, which activates the calcium-activated protease calpain by increasing Ca(2+) release from cytoplasmic stores. Fam38A-induced integrin activation is blocked by inhibition of either R-Ras or calpain activity, or by siRNA knockdown of talin, a well-described calpain substrate. This highlights a novel mechanism for integrin activation by Fam38A, utilising calpain and R-Ras signalling from the ER. These data represent the first description of a novel spatial regulator of R-Ras, of an alternative integrin activation-suppression pathway based on direct relocalisation of R-Ras to the ER, and of a mechanism linking R-Ras and calpain signalling from the ER with modulation of integrin-ligand affinity.

Title: A Viral microRNA Cluster Strongly Potentiates the Transforming Properties of a Human Herpesvirus Content: Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

Title: Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity. Content: Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2(-/-) TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.

Title: Circadian mechanisms in murine and human bone marrow mesenchymal stem cells following dexamethasone exposure. Content: A core group of regulatory factors control circadian rhythms in mammalian cells. While the suprachiasmatic nucleus in the brain serves as the central core circadian oscillator, circadian clocks also exist within peripheral tissues and cells. A growing body of evidence has demonstrated that >20% of expressed mRNAs in bone and adipose tissues oscillate in a circadian manner. The current manuscript reports evidence of the core circadian transcriptional apparatus within primary cultures of murine and human bone marrow-derived mesenchymal stem cells (BMSCs). Exposure of confluent, quiescent BMSCs to dexamethasone synchronized the oscillating expression of the mRNAs encoding the albumin D binding protein (dbp), brain-muscle arnt-like 1 (bmal1), period 3 (per3), rev-erb alpha (Rev A), and rev-erb beta (Rev B). The genes displayed a mean oscillatory period of 22.2 to 24.3 h. The acrophase or peak expression of mRNAs encoding "positive" (bmal1) and "negative" (per3) components of the circadian regulatory apparatus were out of phase with each other by approximately 8-12 h, consistent with in vivo observations. In vivo, phosphyrylation by glycogen synthase kinase 3beta (GSK3beta) is known to regulate the turnover of per3 and components of the core circadian regulatory apparatus. In vitro addition of lithium chloride, a GSK3beta inhibitor, significantly shifted the acrophase of all genes by 4.2-4.7 h oscillation in BMSCs; however, only the male murine BMSCs displayed a significant increase in the length of the period of oscillation. We conclude that human and murine BMSCs represent a valid in vitro model for the analysis of circadian mechanisms in bone metabolism and stem cell biology.

Title: Innovative beneficial reuse of reverse osmosis concentrate using bipolar membrane electrodialysis and electrochlorination processes Content: Reverse osmosis (RO) is a widely used and rapidly growing desalination technology. A major disadvantage of this process is that the concentrate from the RO process, which could be as much as 25% of the feed stream, represents a polluting stream. This waste stream could pose a significant challenge to the implementation of this process, particularly for inland communities which do not have the option of ocean disposal. An excellent environmentally benign approach to disposal could be beneficial reuse of the waste stream. This study presents two innovative beneficial reuse strategies for RO concentrate produced by an integrated membrane system (IMS) from a wastewater reclamation facility. The technologies evaluated in this study included bipolar membrane electrodialysis (BMED) for conversion of RO concentrate into mixed acid and mixed base streams, and electrochlorination (EC) for onsite chlorine generation. Bench-scale studies conducted with BMED demonstrated that RO concentrate could be desalted while producing mixed acids and mixed bases with concentrations as high as 0.2N. Similarly, the EC process was capable of producing a 0.6% hypochlorite solution from RO concentrate. The acids and bases as well as the hypochlorite produced could be directly applied to the RO process as well as upstream pre-treatment processes. A preliminary economic evaluation of the viability of these two approaches was conducted by conducting rough order of magnitude cost estimates based on the bench-scale performance of these processes on RO concentrate. A comparison of the overall costs of an Integrated Membrane System utilizing these innovative reuse strategies with conventional disposal options and thermal zero liquid discharge treatment is presented. This comparison indicates that a reuse approach might be economically viable for inland wastewater reuse facilities that utilize RO membranes and have limited options for concentrate disposal.

Title: Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors. Content: We examined the effects of an inhibitor of PI3K, XL147, against human breast cancer cell lines with constitutive PI3K activation. Treatment with XL147 resulted in dose-dependent inhibition of cell growth and levels of pAKT and pS6, signal transducers in the PI3K/AKT/TOR pathway. In HER2-overexpressing cells, inhibition of PI3K was followed by up-regulation of expression and phosphorylation of multiple receptor tyrosine kinases, including HER3. Knockdown of FoxO1 and FoxO3a transcription factors suppressed the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon inhibition of PI3K. In HER2(+) cells, knockdown of HER3 with siRNA or cotreatment with the HER2 inhibitors trastuzumab or lapatinib enhanced XL147-induced cell death and inhibition of pAKT and pS6. Trastuzumab and lapatinib each synergized with XL147 for inhibition of pAKT and growth of established BT474 xenografts. These data suggest that PI3K antagonists will inhibit AKT and relieve suppression of receptor tyrosine kinase expression and their activity. Relief of this feedback limits the sustained inhibition of the PI3K/AKT pathway and attenuates the response to these agents. As a result, PI3K pathway inhibitors may have limited clinical activity overall if used as single agents. In patients with HER2-overexpressing breast cancer, PI3K inhibitors should be used in combination with HER2/HER3 antagonists.

Title: Neuregulin 1 promotes excitatory synapse development and function in GABAergic interneurons. Content: Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD-95 puncta and the frequency and amplitude of miniature EPSCs (mEPSCs) in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced frequency and amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in excitatory synaptogenesis in interneurons. Together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.

Title: ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast Content: Mitochondrial division is important for mitochondrial distribution and function. Recent data have demonstrated that ER-mitochondria contacts mark mitochondrial division sites, but the molecular basis and functions of these contacts are not understood. Here we show that in yeast, the ER-mitochondria tethering complex, ERMES, and the highly conserved Miro GTPase, Gem1, are spatially and functionally linked to ER-associated mitochondrial division. Gem1 acts as a negative regulator of ER-mitochondria contacts, an activity required for the spatial resolution and distribution of newly generated mitochondrial tips following division. Previous data have demonstrated that ERMES localizes with a subset of actively replicating mitochondrial nucleoids. We show that mitochondrial division is spatially linked to nucleoids and that a majority of these nucleoids segregate prior to division, resulting in their distribution into newly generated tips in the mitochondrial network. Thus, we postulate that ER-associated division serves to link the distribution of mitochondria and mitochondrial nucleoids in cells. DOI:http://dx.doi.org/10.7554/eLife.00422.001.

Title: Regulation of ADMP and BMP2/4/7 at Opposite Embryonic Poles Generates a Self-Regulating Morphogenetic Field Content: Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.

Title: Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. Content: Combinatorial transcription factor (TF) interactions control cellular phenotypes and, therefore, underpin stem cell formation, maintenance, and differentiation. Here, we report the genome-wide binding patterns and combinatorial interactions for ten key regulators of blood stem/progenitor cells (SCL/TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1, and GFI1B), thus providing the most comprehensive TF data set for any adult stem/progenitor cell type to date. Genome-wide computational analysis of complex binding patterns, followed by functional validation, revealed the following: first, a previously unrecognized combinatorial interaction between a heptad of TFs (SCL, LYL1, LMO2, GATA2, RUNX1, ERG, and FLI-1). Second, we implicate direct protein-protein interactions between four key regulators (RUNX1, GATA2, SCL, and ERG) in stabilizing complex binding to DNA. Third, Runx1(+/-)::Gata2(+/-) compound heterozygous mice are not viable with severe hematopoietic defects at midgestation. Taken together, this study demonstrates the power of genome-wide analysis in generating novel functional insights into the transcriptional control of stem and progenitor cells.

Title: Nuclear Factor-�B Affects Tumor Progression in a Mouse Model of Malignant Pleural Effusion Content: We developed a novel mouse model of malignant pleural effusion (MPE) by injecting Lewis lung cancer (LLC) cells directly into the pleural space of syngeneic C57B/6 mice. The pleural effusions in this model share common cellular and biochemical features with human MPEs. Implantation and growth of pleural tumors triggers a host inflammatory response characterized by a mixed inflammatory cell influx into the pleural fluid. LLC cells exhibited high basal nuclear factor (NF)-κB activity in vitro and in vivo, which we used to drive expression of a NF-κB–dependent green fluorescent protein-firefly luciferase fusion reporter construct. NF-κB–dependent reporter expression allowed intravital tracing of pleural tumors. Inhibition of NF-κB in LLC cells did not affect cell viability in culture; however, injection of LLC cells expressing a dominant NF-κB inhibitor resulted in decreased tumor burden, decreased pleural effusion volume, and decreased pleural effusion TNF-α levels. These studies indicate that tumor NF-κB a...

Title: BMP/SMAD1 signaling sets a threshold for the left/right pathway in lateral plate mesoderm and limits availability of SMAD4. Content: Bistability in developmental pathways refers to the generation of binary outputs from graded or noisy inputs. Signaling thresholds are critical for bistability. Specification of the left/right (LR) axis in vertebrate embryos involves bistable expression of transforming growth factor beta (TGFbeta) member NODAL in the left lateral plate mesoderm (LPM) controlled by feed-forward and feedback loops. Here we provide evidence that bone morphogenetic protein (BMP)/SMAD1 signaling sets a repressive threshold in the LPM essential for the integrity of LR signaling. Conditional deletion of Smad1 in the LPM led to precocious and bilateral pathway activation. NODAL expression from both the left and right sides of the node contributed to bilateral activation, indicating sensitivity of mutant LPM to noisy input from the LR system. In vitro, BMP signaling inhibited NODAL pathway activation and formation of its downstream SMAD2/4-FOXH1 transcriptional complex. Activity was restored by overexpression of SMAD4 and in embryos, elevated SMAD4 in the right LPM robustly activated LR gene expression, an effect reversed by superactivated BMP signaling. We conclude that BMP/SMAD1 signaling sets a bilateral, repressive threshold for NODAL-dependent Nodal activation in LPM, limiting availability of SMAD4. This repressive threshold is essential for bistable output of the LR system.

Title: Fast and accurate short read alignment with Burrows–Wheeler transform Content: MOTIVATION The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. RESULTS We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows-Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is approximately 10-20x faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. AVAILABILITY http://maq.sourceforge.net.

Title: Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase Content: During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Title: Endothelial-myofibroblast transition contributes to the early development of diabetic renal interstitial fibrosis in streptozotocin-induced diabetic mice. Content: Diabetic nephropathy is the leading cause of chronic renal failure. Myofibroblasts play a major role in the synthesis and secretion of extracellular matrix in diabetic renal fibrosis. Increasing evidence suggests that endothelial cells may undergo endothelial-myofibroblast transition under physiological and pathophysiological circumstances. Therefore, this study investigates whether endothelial-myofibroblast transition occurs and contributes to the development of diabetic renal interstitial fibrosis. Diabetes was induced by administration of streptozotocin to Tie2-Cre;LoxP-EGFP mice, an endothelial lineage-traceable mouse line generated by crossbreeding B6.Cg-Tg(Tek-cre)12F1v/J mice with B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J mice. The endothelial-myofibroblast transition was also studied in MMECs (a mouse pancreatic microvascular endothelial cell line) and primary cultures of CD31+/EYFP- (enhanced yellow fluorescent protein) endothelial cells isolated from adult normal alpha-smooth muscle actin promoter-driven-EYFP (alpha-SMA/EYFP) mouse kidneys. Confocal microscopy demonstrated that 10.4 +/- 4.2 and 23.5 +/- 7.4% of renal interstitial myofibroblasts (alpha-SMA+) in 1- and 6-month streptozotocin-induced diabetic kidneys were of endothelial origin (EGFP+/alpha-SMA+ cells), compared with just 0.2 +/- 0.1% of myofibroblasts in vehicle-treated Tie2-Cre;LoxP-EGFP mice (P < 0.01). Confocal microscopy and real-time PCR showed that transforming growth factor (TGF)-beta1 induced de novo expression of alpha-SMA and loss of expression of VE-cadherin and CD31 in MMECs and primary cultures of renal endothelial cells in a time- and dose-dependent fashion. These findings demonstrate that the endothelial-myofibroblast transition occurs and contributes to the early development and progression of diabetic renal interstitial fibrosis and suggest that the endothelial-myofibroblast transition may be a therapeutic target.

Title: Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions Content: Epidermal homeostasis depends on a balance between stem cell renewal and differentiation and is regulated by extrinsic signals from the extracellular matrix (ECM). A powerful approach to analysing the pathways involved is to engineer single-cell microenvironments in which individual variables are precisely and quantitatively controlled. Here, we employ micropatterned surfaces to identify the signalling pathways by which restricted ECM contact triggers human epidermal stem cells to initiate terminal differentiation. On small (20 μm diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50 μm diameter) islands. Differentiation did not depend on ECM composition or density. Rather, the actin cytoskeleton mediated shape-induced differentiation by regulating serum response factor (SRF) transcriptional activity. Knockdown of SRF or its co-factor MAL inhibited differentiation, whereas overexpression of MAL stimulated SRF activity and involucrin expression. SRF target genes FOS and JUNB were also required for differentiation: c-Fos mediated serum responsiveness, whereas JunB was regulated by actin and MAL. Our findings demonstrate how biophysical cues are transduced into transcriptional responses that determine epidermal cell fate.

Title: CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappaB subunits on IL-8 and Bcl-xL gene promoters. Content: CD28 is one of the most important costimulatory receptors necessary for full T lymphocyte activation. The CD28 receptor can enhance T cell antigen receptor (TCR) signals, as well as deliver independent signals. Indeed, CD28 engagement by B7 can generate TCR-independent signals leading to IkappaB kinase and NF-kappaB activation. Here we demonstrate that the TCR-independent CD28 signal leads to the selective transcription of survival (Bcl-xL) and inflammatory (IL-8 and B cell activation factor, but not proliferative (IL-2), genes, in a NF-kappaB-dependent manner. CD28-stimulated T cells actively secrete IL-8, and Bcl-xL up-regulation protects T cells from radiation-induced apoptosis. The transcription of CD28-induced genes is mediated by the specific recruitment of RelA and p52 NF-kappaB subunits to target promoters. In contrast, p50 and c-Rel, which preferentially bind NF-kappaB sites on the IL-2 gene promoter after anti-CD3 stimulation, are not involved. Thus, we identify CD28 as a key regulator of genes important for both survival and inflammation.

Title: Follicular B cell trafficking within the spleen actively restricts humoral immune responses. Content: Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen, but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naive FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens.

Title: Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes Content: Increased lipid supply causes beta cell death, which may contribute to reduced beta cell mass in type 2 diabetes. We investigated whether endoplasmic reticulum (ER) stress is necessary for lipid-induced apoptosis in beta cells and also whether ER stress is present in islets of an animal model of diabetes and of humans with type 2 diabetes. Expression of genes involved in ER stress was evaluated in insulin-secreting MIN6 cells exposed to elevated lipids, in islets isolated from db/db mice and in pancreas sections of humans with type 2 diabetes. Overproduction of the ER chaperone heat shock 70 kDa protein 5 (HSPA5, previously known as immunoglobulin heavy chain binding protein [BIP]) was performed to assess whether attenuation of ER stress affected lipid-induced apoptosis. We demonstrated that the pro-apoptotic fatty acid palmitate triggers a comprehensive ER stress response in MIN6 cells, which was virtually absent using non-apoptotic fatty acid oleate. Time-dependent increases in mRNA levels for activating transcription factor 4 (Atf4), DNA-damage inducible transcript 3 (Ddit3, previously known as C/EBP homologous protein [Chop]) and DnaJ homologue (HSP40) C3 (Dnajc3, previously known as p58) correlated with increased apoptosis in palmitate- but not in oleate-treated MIN6 cells. Attenuation of ER stress by overproduction of HSPA5 in MIN6 cells significantly protected against lipid-induced apoptosis. In islets of db/db mice, a variety of marker genes of ER stress were also upregulated. Increased processing (activation) of X-box binding protein 1 (Xbp1) mRNA was also observed, confirming the existence of ER stress. Finally, we observed increased islet protein production of HSPA5, DDIT3, DNAJC3 and BCL2-associated X protein in human pancreas sections of type 2 diabetes subjects. Our results provide evidence that ER stress occurs in type 2 diabetes and is required for aspects of the underlying beta cell failure.

Title: Endosome-ER Contacts Control Actin Nucleation and Retromer Function through VAP-Dependent Regulation of PI4P Content: VAP (VAPA and VAPB) is an evolutionarily conserved endoplasmic reticulum (ER)-anchored protein that helps generate tethers between the ER and other membranes through which lipids are exchanged across adjacent bilayers. Here, we report that by regulating PI4P levels on endosomes, VAP affects WASH-dependent actin nucleation on these organelles and the function of the retromer, a protein coat responsible for endosome-to-Golgi traffic. VAP is recruited to retromer budding sites on endosomes via an interaction with the retromer SNX2 subunit. Cells lacking VAP accumulate high levels of PI4P, actin comets, and trans-Golgi proteins on endosomes. Such defects are mimicked by downregulation of OSBP, a VAP interactor and PI4P transporter that participates in VAP-dependent ER-endosomes tethers. These results reveal a role of PI4P in retromer-/WASH-dependent budding from endosomes. Collectively, our data show how the ER can control budding dynamics and association with the cytoskeleton of another membrane by direct contacts leading to bilayer lipid modifications.

Title: Self-Reactive B Cells in the Germinal Center Reaction. Content: Maintenance of immunological self-tolerance requires lymphocytes carrying self-reactive antigen receptors to be selectively prevented from mounting destructive or inflammatory effector responses. Classically, self-tolerance is viewed in terms of the removal, editing, or silencing of B and T cells that have formed self-reactive antigen receptors during their early development. However, B cells activated by foreign antigen can enter germinal centers (GCs), where they further modify their antigen receptor by somatic hypermutation (SHM) of their immunoglobulin genes. The inevitable emergence of activated, self-reactive GC B cells presents a unique challenge to the maintenance of self-tolerance that must be rapidly countered to avoid autoantibody production. Here we discuss current knowledge of the mechanisms that enforce B cell self-tolerance, with particular focus on the control of self-reactive GC B cells. We also consider how self-reactive GC B cells can escape self-tolerance to initiate autoantibody production or instead be redeemed via SHM and used in productive antibody responses.

Title: The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. Content: The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.

Title: BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium. Content: The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.

Title: Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin. Content: Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.

Title: TLR-activated B cells suppress T cell-mediated autoimmunity. Content: TLR sense microbial infections, and control activation of immune responses. Dendritic cells, macrophages, and B lymphocytes express TLR and the TLR-signaling adaptor protein MyD88. The impact of TLR-activated B cells on T cell-mediated inflammation is unknown. In this study, we have used mice carrying B cell-restricted deficiencies in MyD88 or in distinct TLR to examine the impact of TLR-activated B cells on a T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE). We demonstrate that TLR-signaling in B cells suppresses inflammatory T cell responses (both Th1 and Th17), and stimulates recovery from EAE. Only certain TLR are required on B cells for resolution of EAE, and these are dispensable for disease initiation, indicating that a category of TLR agonists preferentially triggers a suppressive function in B cells and thereby limits autoimmune disease. The TLR agonists controlling the regulatory function of B cells are provided by components of Mycobacterium tuberculosis present in the adjuvant. Thus, MyD88 signaling in B cells antagonizes MyD88 signaling in other cells, which drives differentiation of Th17 cells and is required for induction of EAE. Altogether, our data indicate that B cells link recognition of microbial products via TLR to suppression of a T cell-mediated autoimmune disease.