query: Deletion of αvβ8 does not result in a spontaneous inflammatory phenotype.

positive: Integrin αvβ8-Mediated TGF-β Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation Regulatory T (Treg) cells play a pivotal role in suppressing self-harmful T cell responses, but how Treg cells mediate suppression to maintain immune homeostasis and limit responses during inflammation is unclear. Here we show that effector Treg cells express high amounts of the integrin αvβ8, which enables them to activate latent transforming growth factor-β (TGF-β). Treg-cell-specific deletion of integrin αvβ8 did not result in a spontaneous inflammatory phenotype, suggesting that this pathway is not important in Treg-cell-mediated maintenance of immune homeostasis. However, Treg cells lacking expression of integrin αvβ8 were unable to suppress pathogenic T cell responses during active inflammation. Thus, our results identify a mechanism by which Treg cells suppress exuberant immune responses, highlighting a key role for effector Treg-cell-mediated activation of latent TGF-β in suppression of self-harmful T cell responses during active inflammation.

negative: Characterization of Programmed Death-1 Homologue-1 (PD-1H) Expression and Function in Normal and HIV Infected Individuals Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.

negative: `Gain of function' phenotype of tumor-derived mutant p53 requires the oligomerization/nonsequence-specific nucleic acid-binding domain Tumor-derived p53 mutants can transcriptionally activate a number of promoters of genes involved in cellular proliferation. For this transactivation, mutant p53 does not use the wild-type p53 DNA-binding site, suggesting a mechanism of transactivation that is independent of direct DNA binding. Here we describe our analysis of the domain requirements for mutant p53 to transactivate promoters of the human epidermal growth factor receptor (EGFR), human multiple drug resistance 1 (MDR-1) and human proliferating cell nuclear antigen (PCNA) genes. We also report the identification of a structural domain required for the `gain of function' property of mutant p53-281G. `Gain of function' is measured as the tumorigenicity (in nude mice) of 10(3) murine cells expressing mutant p53 constitutively. We have generated internal deletion mutants of p53-281G deleting conserved domains I, II, III, IV and V, individually. We have also generated one deletion mutant eliminating amino acids 100 through 300 that removes four of the five conserved domains (II–V); another mutant, p53-281G del 393-327, deletes the oligomerization and nonsequence-specific nucleic acid-binding domains of p53. For the EGFR and MDR-1 promoters, all these mutants have significantly lower transactivation ability than intact p53-281G. These deletion mutants, however, significantly activated the pCNA promoter, suggesting that the mechanism of transactivation of the PCNA promoter is different from that of the EGFR and MDR-1 promoters. When expressed constitutively in 10(3) cells, p53-281G del 393-327 was found to be defective in inducing tumor formation in nude mice although intact p53-281G was very efficient. Thus, our results suggest that structural domains near the C-terminus are needed for `gain of function'.

negative: Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus-induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.

negative: Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception. Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH(2) into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1beta concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH(2) evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH(2), injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1beta production are independent of this channel.

negative: Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing. Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ~1 × 10(-3) per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5'ApC3'/3'TpG5' sites (where bases 5'A and 3'T are mutated) and, to a lesser extent, at 5'GpC3'/3'CpG5' sites (where bases 5'G and 3'C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.

negative: Spontaneous autoimmunity prevented by thymic expression of a single self-antigen The expression of self-antigen in the thymus is believed to be responsible for the deletion of autoreactive T lymphocytes, a critical process in the maintenance of unresponsiveness to self. The Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. A clear link, however, between specific thymic self-antigens and a single autoimmune phenotype in this model has been lacking. We show that autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals.

negative: Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A (encoded by LMNA), one of the major architectural elements of the mammalian cell nucleus. The HGPS mutation activates an aberrant cryptic splice site in LMNA pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction in wild-type lamin A. Fibroblasts from individuals with HGPS have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from individuals with HGPS. Introduction of wild-type lamin A protein does not rescue the cellular disease symptoms. The mutant LMNA mRNA and lamin A protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the correction of the premature aging phenotype in individuals with HGPS.

negative: CD28 and ITK signals regulate autoreactive T cell trafficking Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

negative: Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein. We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.

negative: Activation of Frontal Neocortical Areas by Vocal Production in Marmosets Primates often rely on vocal communication to mediate social interactions. Although much is known about the acoustic structure of primate vocalizations and the social context in which they are usually uttered, our knowledge about the neocortical control of audio-vocal interactions in primates is still incipient, being mostly derived from lesion studies in squirrel monkeys and macaques. To map the neocortical areas related to vocal control in a New World primate species, the common marmoset, we employed a method previously used with success in other vertebrate species: Analysis of the expression of the immediate early gene Egr-1 in freely behaving animals. The neocortical distribution of Egr-1 immunoreactive cells in three marmosets that were exposed to the playback of conspecific vocalizations and vocalized spontaneously (H/V group) was compared to data from three other marmosets that also heard the playback but did not vocalize (H/n group). The anterior cingulate cortex, the dorsomedial prefrontal cortex and the ventrolateral prefrontal cortex presented a higher number of Egr-1 immunoreactive cells in the H/V group than in H/n animals. Our results provide direct evidence that the ventrolateral prefrontal cortex, the region that comprises Broca's area in humans and has been associated with auditory processing of species-specific vocalizations and orofacial control in macaques, is engaged during vocal output in marmosets. Altogether, our results support the notion that the network of neocortical areas related to vocal communication in marmosets is quite similar to that of Old world primates. The vocal production role played by these areas and their importance for the evolution of speech in primates are discussed.

negative: A study of high cell density cultivation process of recombinant Helicobacter pylori multi-epitope vaccine engineering bacteria. OBJECTIVE To establish high cell density cultivation process of recombinant Helicobacter pylori multi-epitope vaccine engineering bacteria BIB. METHODS Based on the results of shake flask fermentation, the process was magnified into volume of a 50 L fermenter to optimize and verify the factors affecting the yield of the target protein, such as the fermentation medium, working seed inoculation amount, inducer concentration, induction starting time, induction duration, inducer adding mode and feeding strategy. RESULTS After activated in modified TB medium at 37°C for 8 h, the BIB working seed was inoculated at 5% (v/v) and was induced for expression for another 11 h by the final concentration of 5 mmol/L lactose. In growth phase, glucose at rate of 80 ml/h was used as carbon source, and in induction phase, glycerol at rate of 40 ml/h was used as carbon source; ammonia water was added dropwise to control pH at about 7.0, and revolution speed is adjusted to control the dissolved oxygen at above 30%; ultimately the output of bacterial body was 70 g/L and protein expression amount was about 32%. CONCLUSION After high cell density cultivation of the recombinant engineering bacteria, expression and yield of the target protein rBIB significantly increased.

negative: Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect. Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24-deficient mice (Zmpste24(-/-)) have indicated a role for Zmpste24 in the processing of CAAX-type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24(-/-) mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures-akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24(-/-) mice. Zmpste24(-/-) mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24(-/-) mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.

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

negative: Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the β-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus–E. coli plasmid shuttle vectors were constructed, which genetically complemented ΔpyrEFΔlacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.

negative: Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties. It has been proposed that epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features, and that the presence of EMT characteristics in claudin-low breast tumors reveals their origin in basal stem cells. It remains to be determined, however, whether EMT is an inherent property of normal basal stem cells, and if the presence of a mesenchymal-like phenotype is required for the maintenance of all their stem cell properties. We used nontumorigenic basal cell lines as models of normal stem cells/progenitors and demonstrate that these cell lines contain an epithelial subpopulation ("EpCAM+," epithelial cell adhesion molecule positive [EpCAM(pos)]/CD49f(high)) that spontaneously generates mesenchymal-like cells ("Fibros," EpCAM(neg)/CD49f(med/low)) through EMT. Importantly, stem cell/progenitor properties such as regenerative potential, high aldehyde dehydrogenase 1 activity, and formation of three-dimensional acini-like structures predominantly reside within EpCAM+ cells, while Fibros exhibit invasive behavior and mammosphere-forming ability. A gene expression profiling meta-analysis established that EpCAM+ cells show a luminal progenitor-like expression pattern, while Fibros most closely resemble stromal fibroblasts but not stem cells. Moreover, Fibros exhibit partial myoepithelial traits and strong similarities with claudin-low breast cancer cells. Finally, we demonstrate that Slug and Zeb1 EMT-inducers control the progenitor and mesenchymal-like phenotype in EpCAM+ cells and Fibros, respectively, by inhibiting luminal differentiation. In conclusion, nontumorigenic basal cell lines have intrinsic capacity for EMT, but a mesenchymal-like phenotype does not correlate with the acquisition of global stem cell/progenitor features. Based on our findings, we propose that EMT in normal basal cells and claudin-low breast cancers reflects aberrant/incomplete myoepithelial differentiation.

negative: Loss of allergen 1 confers a hypervirulent phenotype that resembles mucoid switch variants of Cryptococcus neoformans. Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Delta gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Delta cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Delta mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function.

negative: Inflammatory Reaction as Determinant of Foreign Body Reaction Is an Early and Susceptible Event after Mesh Implantation PURPOSE To investigate and relate the ultrashort-term and long-term courses of determinants for foreign body reaction as biocompatibility predictors for meshes in an animal model. MATERIALS AND METHODS Three different meshes (TVT, UltraPro, and PVDF) were implanted in sheep. Native and plasma coated meshes were placed bilaterally: (a) interaperitoneally, (b) as fascia onlay, and (c) as muscle onlay (fascia sublay). At 5 min, 20 min, 60 min, and 120 min meshes were explanted and histochemically investigated for inflammatory infiltrate, macrophage infiltration, vessel formation, myofibroblast invasion, and connective tissue accumulation. The results were related to long-term values over 24 months. RESULTS Macrophage invasion reached highest extents with up to 60% in short-term and decreased within 24 months to about 30%. Inflammatory infiltrate increased within the first 2 hours, the reached levels and the different extents and ranking among the investigated meshes remained stable during long-term follow up. For myofibroblasts, connective tissue, and CD31+ cells, no activity was detected during the first 120 min. CONCLUSION The local inflammatory reaction is an early and susceptible event after mesh implantation. It cannot be influenced by prior plasma coating and does not depend on the localisation of implantation.

negative: Islet1 deletion causes kidney agenesis and hydroureter resembling CAKUT. Islet1 (Isl1) is a transcription factor transiently expressed in a subset of heart and limb progenitors. During studies of limb development, conditional Isl1 deletion produced unexpected kidney abnormalities. Here, we studied the renal expression of Isl1 and whether it has a role in kidney development. In situ hybridization revealed Isl1 expression in the mesenchymal cells surrounding the base of the ureteric bud in mice. Conditional deletion of Isl1 caused kidney agenesis or hypoplasia and hydroureter, a phenotype resembling human congenital anomalies of the kidney and urinary tract (CAKUT). The absence of Isl1 led to ectopic branching of the ureteric bud out from the nephric duct or to the formation of accessory buds, both of which could lead to obstruction of the ureter-bladder junction and consequent hydroureter. The abnormal elongation and poor branching of the ureteric buds were the likely causes of the kidney agenesis or hypoplasia. Furthermore, the lack of Isl1 reduced the expression of Bmp4, a gene implicated in the CAKUT-like phenotype, in the metanephric region before ureteric budding. In conclusion, Isl1 is essential for proper development of the kidney and ureter by repressing the aberrant formation of the ureteric bud. These observations call for further studies to investigate whether Isl1 may be a causative gene for human CAKUT.

negative: Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation. Although 75% of endometrial cancers are treated at an early stage, 15% to 20% of these recur. We performed an integrated analysis of genome-wide expression and copy-number data for primary endometrial carcinomas with extensive clinical and histopathological data to detect features predictive of recurrent disease. Unsupervised analysis of the expression data distinguished 2 major clusters with strikingly different phenotypes, including significant differences in disease-free survival. To identify possible mechanisms for these differences, we performed a global genomic survey of amplifications, deletions, and loss of heterozygosity, which identified 11 significantly amplified and 13 significantly deleted regions. Amplifications of 3q26.32 harboring the oncogene PIK3CA were associated with poor prognosis and segregated with the aggressive transcriptional cluster. Moreover, samples with PIK3CA amplification carried signatures associated with in vitro activation of PI3 kinase (PI3K), a signature that was shared by aggressive tumors without PIK3CA amplification. Tumors with loss of PTEN expression or PIK3CA overexpression that did not have PIK3CA amplification also shared the PI3K activation signature, high protein expression of the PI3K pathway member STMN1, and an aggressive phenotype in test and validation datasets. However, mutations of PTEN or PIK3CA were not associated with the same expression profile or aggressive phenotype. STMN1 expression had independent prognostic value. The results affirm the utility of systematic characterization of the cancer genome in clinically annotated specimens and suggest the particular importance of the PI3K pathway in patients who have aggressive endometrial cancer.

negative: The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways. The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

negative: Localization of Na+ channel isoforms at the atrioventricular junction and atrioventricular node in the rat. BACKGROUND The electrical activity of the atrioventricular node (AVN) is functionally heterogeneous, but how this relates to distinct cell types and the 3-dimensional structure of the AVN is unknown. To address this, we have studied the expression of Na(V)1.5 and other Na+ channel isoforms in the AVN. METHODS AND RESULTS The rat AVN was identified by Masson's trichrome staining together with immunolabeling of marker proteins: connexin40, connexin43, desmoplakin, atrial natriuretic peptide, and hyperpolarization-activated and cyclic nucleotide-gated channel 4. Na+ channel expression was investigated with immunohistochemistry with isoform-specific Na+ channel antibodies. Na(V)1.1 was distributed in a similar manner to Na(V)1.5. Na(V)1.2 was not detected. Na(V)1.3 labeling was present in nerve fibers and cell bodies (but not myocytes) and was abundant in the penetrating atrioventricular (AV) bundle and the common bundle but was much less abundant in other regions. Na(V)1.5 labeling was abundant in the atrial and ventricular myocardium and the left bundle branch. Na(V)1.5 labeling was absent in the open node, penetrating AV bundle, AV ring bundle, and common bundle but present at a reduced level in the inferior nodal extension and transitional zone. Na(V)1.6 was not detected. CONCLUSIONS Our findings provide molecular evidence of multiple electrophysiological cell types at the AV junction. Impaired AV conduction as a result of mutations in or loss of Na(V)1.5 must be the result of impaired conduction in the AVN inputs (inferior nodal extension and transitional zone) or output (bundle branches) rather than the AVN itself (open node and penetrating AV bundle).

negative: Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

negative: Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer Antigen-specific CD8+ T-cell tolerance, induced by myeloid-derived suppressor cells (MDSCs), is one of the main mechanisms of tumor escape. Using in vivo models, we show here that MDSCs directly disrupt the binding of specific peptide–major histocompatibility complex (pMHC) dimers to CD8-expressing T cells through nitration of tyrosines in a T-cell receptor (TCR)-CD8 complex. This process makes CD8-expressing T cells unable to bind pMHC and to respond to the specific peptide, although they retain their ability to respond to nonspecific stimulation. Nitration of TCR-CD8 is induced by MDSCs through hyperproduction of reactive oxygen species and peroxynitrite during direct cell-cell contact. Molecular modeling suggests specific sites of nitration that might affect the conformational flexibility of TCR-CD8 and its interaction with pMHC. These data identify a previously unknown mechanism of T-cell tolerance in cancer that is also pertinent to many pathological conditions associated with accumulation of MDSCs.

negative: YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance Previous results have demonstrated that the silencing of adjacent genes encoding NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ Microbiol 75:4315–4323, 2009]. This gene silencing is now reported to result from the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream gene. YqhC shares homology with transcriptional regulators belonging to the AraC/XylS family and was shown to act as a positive regulator of the adjacent operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and by a direct comparison of furfural resistance and NADPH-dependent furfural reductase activity. Closely related bacteria contain yqhC, yqhD, and dkgA orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also present in more distantly related Gram-negative bacteria. Disruption of yqhC offers a useful approach to increase furfural tolerance in bacteria.

negative: Hypoxia‐Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis Summary Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell‐intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF‐&kgr;B signaling and a transcription factor E2F1‐dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL‐induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine‐driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F‐metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction. Graphical Abstract Figure. No Caption available. HighlightsCOMMD1 is a negative regulator of osteoclast differentiationCOMMD1 suppresses bone loss in RA and inflammatory arthritis and osteolysis modelsCOMMD1 negatively regulates E2F1‐dependent metabolic pathways in macrophagesHypoxia suppresses COMMD1 expression to augment osteoclastogenesis &NA; Pathways that promote osteoclastogenesis are well characterized but less is known about negative regulators that suppress pathological bone loss. Murata et al. identify COMMD1 as an inhibitor of osteoclastogenesis that restrains NF‐&kgr;B‐ and E2F1‐CKB‐mediated metabolic pathways in macrophages.

negative: miR-302 Is Required for Timing of Neural Differentiation, Neural Tube Closure, and Embryonic Viability The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development.

negative: KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis Previous studies investigating the role of smooth muscle cells (SMCs) and macrophages in the pathogenesis of atherosclerosis have provided controversial results owing to the use of unreliable methods for clearly identifying each of these cell types. Here, using Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− mice to perform SMC lineage tracing, we find that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages, including macrophages and mesenchymal stem cells (MSCs). SMC-specific conditional knockout of Krüppel-like factor 4 (Klf4) resulted in reduced numbers of SMC-derived MSC- and macrophage-like cells, a marked reduction in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness as compared to wild-type controls. On the basis of in vivo KLF4 chromatin immunoprecipitation–sequencing (ChIP-seq) analyses and studies of cholesterol-treated cultured SMCs, we identified >800 KLF4 target genes, including many that regulate pro-inflammatory responses of SMCs. Our findings indicate that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.

negative: Local neuroinflammation and the progression of Alzheimer’s disease Postmortem immunohistochemical studies have revealed a state of chronic inflammation limited to lesioned areas of brain in Alzheimer’s disease. Some key actors in this inflammation are activated microglia (brain macrophages), proteins of the classical complement cascade, the pentraxins, cytokines, and chemokines. The inflammation does not involve the adaptive immune system or peripheral organs, but is rather due to the phylogenetically much older innate immune system, which appears to operate in most tissues of the body. Chronic inflammation can damage host tissue and the brain may be particularly vulnerable because of the postmitotic nature of neurons. Many of the inflammatory mediators have been shown to be locally produced and selectively elevated in affected regions of Alzheimer’s brain. Moreover, studies of tissue in such degenerative processes as atherosclerosis and infarcted heart suggest a similar local innate immune reaction may be important in such conditions. Much epidemiological and limited clinical evidence suggests that nonsteroidal anti-inflammatory drugs may impede the onset and slow the progression of Alzheimer’s disease. But these drugs strike at the periphery of the inflammatory reaction. Much better results might be obtained if drugs were found that could inhibit the activation of microglia or the complement system in brain, and combinations of drugs aimed at different inflammatory targets might be much more effective than single agents.

negative: Mechanism of NO-mediated oxidative and nitrative DNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis. Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.

negative: Bacteriophage latent-period evolution as a response to resource availability. Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models--in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy--can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (approximately 10(8) cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (10(7) cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 10(8) cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 10(5) cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small.

negative: TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis. The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

negative: TLR-activated B cells suppress T cell-mediated autoimmunity. 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.


query: Gene expression does not vary appreciably across genetically identical cells.

positive: Nature, Nurture, or Chance: Stochastic Gene Expression and Its Consequences Gene expression is a fundamentally stochastic process, with randomness in transcription and translation leading to cell-to-cell variations in mRNA and protein levels. This variation appears in organisms ranging from microbes to metazoans, and its characteristics depend both on the biophysical parameters governing gene expression and on gene network structure. Stochastic gene expression has important consequences for cellular function, being beneficial in some contexts and harmful in others. These situations include the stress response, metabolism, development, the cell cycle, circadian rhythms, and aging.

positive: Memory and Modularity in Cell-Fate Decision Making Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. We show that the motile state is 'memoryless', exhibiting no autonomous control over the time spent in the state. In contrast, the time spent as connected chains of cells is tightly controlled, enforcing coordination among related cells in the multicellular state. We show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.

negative: The Mammalian Epigenome Chemical modifications to DNA and histone proteins form a complex regulatory network that modulates chromatin structure and genome function. The epigenome refers to the complete description of these potentially heritable changes across the genome. The composition of the epigenome within a given cell is a function of genetic determinants, lineage, and environment. With the sequencing of the human genome completed, investigators now seek a comprehensive view of the epigenetic changes that determine how genetic information is made manifest across an incredibly varied background of developmental stages, tissue types, and disease states. Here we review current research efforts, with an emphasis on large-scale studies, emerging technologies, and challenges ahead.

negative: Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells. In this study, we used whole-genome sequencing and gene expression profiling of 215 human induced pluripotent stem cell (iPSC) lines from different donors to identify genetic variants associated with RNA expression for 5,746 genes. We were able to predict causal variants for these expression quantitative trait loci (eQTLs) that disrupt transcription factor binding and validated a subset of them experimentally. We also identified copy-number variant (CNV) eQTLs, including some that appear to affect gene expression by altering the copy number of intergenic regulatory regions. In addition, we were able to identify effects on gene expression of rare genic CNVs and regulatory single-nucleotide variants and found that reactivation of gene expression on the X chromosome depends on gene chromosomal position. Our work highlights the value of iPSCs for genetic association analyses and provides a unique resource for investigating the genetic regulation of gene expression in pluripotent cells.

negative: Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation. RATIONALE Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. OBJECTIVE We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. METHODS AND RESULTS We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. CONCLUSIONS The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

negative: Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division Many stem cells, including Drosophila germline stem cells (GSCs), divide asymmetrically, producing one stem cell and one differentiating daughter. Cytokinesis is often asymmetric, in that only one daughter cell inherits the midbody ring (MR) upon completion of abscission even in apparently symmetrically dividing cells. However, whether the asymmetry in cytokinesis correlates with cell fate or has functional relevance has been poorly explored. Here we show that the MR is asymmetrically segregated during GSC divisions in a centrosome age-dependent manner: male GSCs, which inherit the mother centrosome, exclude the MR, whereas female GSCs, which we here show inherit the daughter centrosome, inherit the MR. We further show that stem cell identity correlates with the mode of MR inheritance. Together our data suggest that the MR does not inherently dictate stem cell identity, although its stereotypical inheritance is under the control of stemness and potentially provides a platform for asymmetric segregation of certain factors.

negative: MicroRNAs can generate thresholds in target gene expression MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We find that although the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically. In particular, we show that regulation by miRNAs establishes a threshold level of target mRNA below which protein production is highly repressed. Near this threshold, protein expression responds sensitively to target mRNA input, consistent with a mathematical model of molecular titration. These results show that miRNAs can act both as a switch and as a fine-tuner of gene expression.

negative: Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite's surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.

negative: A fine-scale map of recombination rates and hotspots across the human genome. Genetic maps, which document the way in which recombination rates vary over a genome, are an essential tool for many genetic analyses. We present a high-resolution genetic map of the human genome, based on statistical analyses of genetic variation data, and identify more than 25,000 recombination hotspots, together with motifs and sequence contexts that play a role in hotspot activity. Differences between the behavior of recombination rates over large (megabase) and small (kilobase) scales lead us to suggest a two-stage model for recombination in which hotspots are stochastic features, within a framework in which large-scale rates are constrained.

negative: All dosage compensation is local: Gene-by-gene regulation of sex-biased expression on the chicken Z chromosome Recent reports have suggested that birds lack a mechanism of wholesale dosage compensation for the Z sex chromosome. This discovery was rather unexpected, as all other animals investigated with chromosomal mechanisms of sex determination have some method to counteract the effects of gene dosage of the dominant sex chromosome in males and females. Despite the lack of a global mechanism of avian dosage compensation, the pattern of gene expression difference between males and females varies a great deal for individual Z-linked genes. This suggests that some genes may be individually dosage compensated, and that some less-than-global pattern of dosage compensation, such as local or temporal, exists on the avian Z chromosome. We used global gene expression profiling in males and females for both somatic and gonadal tissue at several time points in the life cycle of the chicken to assess the pattern of sex-biased gene expression on the Z chromosome. Average fold-change between males and females varied somewhat among tissue time-point combinations, with embryonic brain samples having the smallest gene dosage effects, and adult gonadal tissue having the largest degree of male bias. Overall, there were no neighborhoods of overall dosage compensation along the Z. Taken together, this suggests that dosage compensation is regulated on the Z chromosome entirely on a gene-by-gene level, and can vary during the life cycle and by tissue type. This regulation may be an indication of how critical a given gene's functionality is, as the expression level for essential genes will be tightly regulated in order to avoid perturbing important pathways and networks with differential expression levels in males and females.

negative: Attention-deficit hyperactivity disorder: a category or a continuum? Genetic analysis of a large-scale twin study. OBJECTIVE To investigate heritability and continuum versus categorical approaches to attention-deficit hyperactivity disorder (ADHD), using a large-scale twin sample. METHOD A cohort of 1,938 families with twins and siblings aged 4 to 12 years, recruited from the Australian National Health and Medical Research Council Twin Registry, was assessed for ADHD using a DSM-III-R-based maternal rating scale. Probandwise concordance rates and correlations in monozygotic and dizygotic twins and siblings were calculated, and heritability was examined using the De Fries and Fulker regression technique. RESULTS There was a narrow (additive) heritability of 0.75 to 0.91 which was robust across familial relationships (twin, sibling, and twin-sibling) and across definitions of ADHD as part of a continuum or as a disorder with various symptom cutoffs. There was no evidence for nonadditive genetic variation or for shared family environmental effects. CONCLUSIONS These findings suggest that ADHD is best viewed as the extreme of a behavior that varies genetically throughout the entire population rather than as a disorder with discrete determinants. This has implications for the classification of ADHD and for the identification of genes for this behavior, as well as implications for diagnosis and treatment.

negative: An Expanded Oct4 Interaction Network: Implications for Stem Cell Biology, Development, and Disease The transcription factor Oct4 is key in embryonic stem cell identity and reprogramming. Insight into its partners should illuminate how the pluripotent state is established and regulated. Here, we identify a considerably expanded set of Oct4-binding proteins in mouse embryonic stem cells. We find that Oct4 associates with a varied set of proteins including regulators of gene expression and modulators of Oct4 function. Half of its partners are transcriptionally regulated by Oct4 itself or other stem cell transcription factors, whereas one-third display a significant change in expression upon cell differentiation. The majority of Oct4-associated proteins studied to date show an early lethal phenotype when mutated. A fraction of the human orthologs is associated with inherited developmental disorders or causative of cancer. The Oct4 interactome provides a resource for dissecting mechanisms of Oct4 function, enlightening the basis of pluripotency and development, and identifying potential additional reprogramming factors.

negative: The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways. The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

negative: Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to "hedge their bets" in responding to an uncertain future, and to balance growth and survival in an unpredictable environment.

negative: A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed "bivalent domains," consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.

negative: Effective population size and patterns of molecular evolution and variation The effective size of a population, Ne, determines the rate of change in the composition of a population caused by genetic drift, which is the random sampling of genetic variants in a finite population. Ne is crucial in determining the level of variability in a population, and the effectiveness of selection relative to drift. This article reviews the properties of Ne in a variety of different situations of biological interest, and the factors that influence it. In particular, the action of selection means that Ne varies across the genome, and advances in genomic techniques are giving new insights into how selection shapes Ne.

negative: The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Studies of the mutant gene in Huntington's disease, and for eight related neurodegenerative disorders, have identified polyglutamine (polyQ) expansions as a basis for cellular toxicity. This finding has led to a disease hypothesis that protein aggregation and cellular dysfunction can occur at a threshold of approximately 40 glutamine residues. Here, we test this hypothesis by expression of fluorescently tagged polyQ proteins (Q29, Q33, Q35, Q40, and Q44) in the body wall muscle cells of Caenorhabditis elegans and show that young adults exhibit a sharp boundary at 35-40 glutamines associated with the appearance of protein aggregates and loss of motility. Surprisingly, genetically identical animals expressing near-threshold polyQ repeats exhibited a high degree of variation in the appearance of protein aggregates and cellular toxicity that was dependent on repeat length and exacerbated during aging. The role of genetically determined aging pathways in the progression of age-dependent polyQ-mediated aggregation and cellular toxicity was tested by expressing Q82 in the background of age-1 mutant animals that exhibit an extended lifespan. We observed a dramatic delay of polyQ toxicity and appearance of protein aggregates. These data provide experimental support for the threshold hypothesis of polyQ-mediated toxicity in an experimental organism and emphasize the importance of the threshold as a point at which genetic modifiers and aging influence biochemical environment and protein homeostasis in the cell.

negative: The Neurobiology of Sleep: Genetics, cellular physiology and subcortical networks To appreciate the neural underpinnings of sleep, it is important to view this universal mammalian behaviour at multiple levels of its biological organization. Molecularly, the circadian rhythm of sleep involves interlocking positive- and negative-feedback mechanisms of circadian genes and their protein products in cells of the suprachiasmatic nucleus that are entrained to ambient conditions by light. Circadian information is integrated with information on homeostatic sleep need in nuclei of the anterior hypothalamus. These nuclei interact with arousal systems in the posterior hypothalamus, basal forebrain and brainstem to control sleep onset. During sleep, an ultradian oscillator in the mesopontine junction controls the regular alternation of rapid eye movement (REM) and non-REM sleep. Sleep cycles are accompanied by neuromodulatory influences on forebrain structures that influence behaviour, consciousness and cognition.

negative: Measuring Absolute RNA Copy Numbers at High Temporal Resolution Reveals Transcriptome Kinetics in Development. Transcript regulation is essential for cell function, and misregulation can lead to disease. Despite technologies to survey the transcriptome, we lack a comprehensive understanding of transcript kinetics, which limits quantitative biology. This is an acute challenge in embryonic development, where rapid changes in gene expression dictate cell fate decisions. By ultra-high-frequency sampling of Xenopus embryos and absolute normalization of sequence reads, we present smooth gene expression trajectories in absolute transcript numbers. During a developmental period approximating the first 8 weeks of human gestation, transcript kinetics vary by eight orders of magnitude. Ordering genes by expression dynamics, we find that "temporal synexpression" predicts common gene function. Remarkably, a single parameter, the characteristic timescale, can classify transcript kinetics globally and distinguish genes regulating development from those involved in cellular metabolism. Overall, our analysis provides unprecedented insight into the reorganization of maternal and embryonic transcripts and redefines our ability to perform quantitative biology.

negative: Dscam and DSCAM: complex genes in simple animals, complex animals yet simple genes. Cadherins and the immunoglobulin (Ig) proteins give rise to a multitude of surface receptors, which function as diverse cell adhesion molecules (CAMs) or signal-transducing receptors. These functions are often interdependent, and rely on a high degree of specificity in homophilic binding as well as heterophilic interactions. The Drosophila receptor Dscam is an exceptional example of homophilic binding specificity involved in a number of important biological processes, such as neural wiring and innate immunity. Combinatorial use of alternatively spliced Ig-domains enables the generation of an estimated 18,000 isoform-specific homophilic receptor pairs. Although isoform diversity of Dscam is unique to arthropods, recent genetic analysis of vertebrate DSCAM (Down Syndrome Cell Adhesion Molecule) genes has revealed an intriguing conservation of molecular functions underlying neural wiring. This review covers the multiple functions of Dscam across different species highlighting its remarkable versatility as well as its conserved basic functions in neural development. We discuss how an unprecedented expansion of complex alternative splicing has been uniquely employed by arthropods to generate diverse surface receptors, important for cell-cell communication, molecular self-recognition in neurons, and innate immune defenses. We end with a speculative hypothesis reconciling the striking differences in Dscam and DSCAM gene structures with their conserved functions in molecular recognition underlying neural circuit formation.

negative: Redefining the Genetic Hierarchies Controlling Skeletal Myogenesis: Pax-3 and Myf-5 Act Upstream of MyoD We analyzed Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 homozygous embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation.

negative: The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve-injured groups, which can be difficult to validate, particularly with respect to the specificity of pain modulation. To circumvent this, we used two outbred lines of rats, which are genetically similar except for being genetically segregated as a result of selective breeding for differences in neuropathic pain hypersensitivity. SerpinA3N, a serine protease inhibitor, was upregulated in the dorsal root ganglia (DRG) after nerve injury, which was further validated for its mouse homolog. Mice lacking SerpinA3N developed more neuropathic mechanical allodynia than wild-type (WT) mice, and exogenous delivery of SerpinA3N attenuated mechanical allodynia in WT mice. T lymphocytes infiltrate the DRG after nerve injury and release leukocyte elastase (LE), which was inhibited by SerpinA3N derived from DRG neurons. Genetic loss of LE or exogenous application of a LE inhibitor (Sivelastat) in WT mice attenuated neuropathic mechanical allodynia. Overall, we reveal a novel and clinically relevant role for a member of the serpin superfamily and a leukocyte elastase and crosstalk between neurons and T cells in the modulation of neuropathic pain.

negative: Somatic and Germline Diversification of a Putative Immunoreceptor within One Phylum: Dscam in Arthropods. Arthropod Dscam, the homologue of the human Down Syndrome cell adhesion molecule, is a receptor used by the nervous and immune systems. Unlike in vertebrates, evolutionary pressure has selected and maintained a vast Dscam diversity of isoforms, known to specifying neuronal identity during the nervous system differentiation. This chapter examines the different modes of Dscam diversification in the context of arthropods' evolution and that of their immune system, where its role is controversial. In the single Dscam gene of insects and crustaceans, mutually exclusive alternative splicing affects three clusters of duplicated exons encoding the variable parts of the receptor. The Dscam gene produces over 10,000 isoforms. In the more basal arthropods such as centipedes, Dscam diversity results from a combination of many germline genes (over 80) with, in about half of those, the possibility of alternative splicing affecting only one exon cluster. In the even more basal arthropods, such as chelicerates, no splicing possibility is detected, but there exist dozens of germline Dscam genes. Compared to controlling the expression of multiple germline genes, the somatic mutually alternative splicing within a single gene may offer a simplified way of expressing a large Dscam repertoire. Expressed by hemocytes, Dscam is considered a phagocytic receptor but is also encountered in solution. More information is necessary about its binding to pathogens, its role in phagocytosis, its possible role in specifying hemocyte identity, its kinetics of expression, and the regulation of its RNA splicing to understand how its diversity is linked to immunity.

negative: Developmental gene networks: a triathlon on the course to T cell identity Cells acquire their ultimate identities by activating combinations of transcription factors that initiate and sustain expression of the appropriate cell type-specific genes. T cell development depends on the progression of progenitor cells through three major phases, each of which is associated with distinct transcription factor ensembles that control the recruitment of these cells to the thymus, their proliferation, lineage commitment and responsiveness to T cell receptor signals, all before the allocation of cells to particular effector programmes. All three phases are essential for proper T cell development, as are the mechanisms that determine the boundaries between each phase. Cells that fail to shut off one set of regulators before the next gene network phase is activated are predisposed to leukaemic transformation.

negative: Molecular and Cellular Approaches for Diversifying and Extending Optogenetics Optogenetic technologies employ light to control biological processes within targeted cells in vivo with high temporal precision. Here, we show that application of molecular trafficking principles can expand the optogenetic repertoire along several long-sought dimensions. Subcellular and transcellular trafficking strategies now permit (1) optical regulation at the far-red/infrared border and extension of optogenetic control across the entire visible spectrum, (2) increased potency of optical inhibition without increased light power requirement (nanoampere-scale chloride-mediated photocurrents that maintain the light sensitivity and reversible, step-like kinetic stability of earlier tools), and (3) generalizable strategies for targeting cells based not only on genetic identity, but also on morphology and tissue topology, to allow versatile targeting when promoters are not known or in genetically intractable organisms. Together, these results illustrate use of cell-biological principles to enable expansion of the versatile fast optogenetic technologies suitable for intact-systems biology and behavior.

negative: Coordinate regulation of the IL-4, IL-13, and IL-5 cytokine cluster in Th2 clones revealed by allelic expression patterns. The cytokines IL-4, IL-13, and IL-5 are markers for the Th2 subset of effector T cells and are often expressed together. These cytokine genes are organized within 140 kb of orthologous DNA in both mouse and human. Using IL-4-expressing CD4+ T cell clones derived from F1 mice, we identified allelic polymorphisms for each of these cytokines and assessed the parental identity of the cytokine mRNAs. Both monoallelic and biallelic expression occurred for each gene and for an additional gene, IL-3, that lies with GM-CSF over 450 kb telomeric on the same chromosome. When coexpressed in T cell clones, IL-4 was expressed from the same allele as IL-13 or IL-5 in 81% of instances. In contrast, there was only 52% concordance of these three cytokines at the allelic level among clones that expressed IL-3. Independent expression of the cytokine alleles occurs commonly in T cells, but the clustered locus encompassing IL-4, IL-13, and IL-5 is subject to coordinate regulation.

negative: Global Assessment of Genomic Regions Required for Growth in Mycobacterium tuberculosis Identifying genomic elements required for viability is central to our understanding of the basic physiology of bacterial pathogens. Recently, the combination of high-density mutagenesis and deep sequencing has allowed for the identification of required and conditionally required genes in many bacteria. Genes, however, make up only a part of the complex genomes of important bacterial pathogens. Here, we use an unbiased analysis to comprehensively identify genomic regions, including genes, domains, and intergenic elements, required for the optimal growth of Mycobacterium tuberculosis, a major global health pathogen. We found that several proteins jointly contain both domains required for optimal growth and domains that are dispensable. In addition, many non-coding regions, including regulatory elements and non-coding RNAs, are critical for mycobacterial growth. Our analysis shows that the genetic requirements for growth are more complex than can be appreciated using gene-centric analysis.

negative: Tissue-specific expression and regulation of sexually dimorphic genes in mice. We report a comprehensive analysis of gene expression differences between sexes in multiple somatic tissues of 334 mice derived from an intercross between inbred mouse strains C57BL/6J and C3H/HeJ. The analysis of a large number of individuals provided the power to detect relatively small differences in expression between sexes, and the use of an intercross allowed analysis of the genetic control of sexually dimorphic gene expression. Microarray analysis of 23,574 transcripts revealed that the extent of sexual dimorphism in gene expression was much greater than previously recognized. Thus, thousands of genes showed sexual dimorphism in liver, adipose, and muscle, and hundreds of genes were sexually dimorphic in brain. These genes exhibited highly tissue-specific patterns of expression and were enriched for distinct pathways represented in the Gene Ontology database. They also showed evidence of chromosomal enrichment, not only on the sex chromosomes, but also on several autosomes. Genetic analyses provided evidence of the global regulation of subsets of the sexually dimorphic genes, as the transcript levels of a large number of these genes were controlled by several expression quantitative trait loci (eQTL) hotspots that exhibited tissue-specific control. Moreover, many tissue-specific transcription factor binding sites were found to be enriched in the sexually dimorphic genes.

negative: The genetics of Mexico recapitulates Native American substructure and affects biomedical traits Mexico harbors great cultural and ethnic diversity, yet fine-scale patterns of human genome-wide variation from this region remain largely uncharacterized. We studied genomic variation within Mexico from over 1000 individuals representing 20 indigenous and 11 mestizo populations. We found striking genetic stratification among indigenous populations within Mexico at varying degrees of geographic isolation. Some groups were as differentiated as Europeans are from East Asians. Pre-Columbian genetic substructure is recapitulated in the indigenous ancestry of admixed mestizo individuals across the country. Furthermore, two independently phenotyped cohorts of Mexicans and Mexican Americans showed a significant association between subcontinental ancestry and lung function. Thus, accounting for fine-scale ancestry patterns is critical for medical and population genetic studies within Mexico, in Mexican-descent populations, and likely in many other populations worldwide.

negative: ALS-FUS pathology revisited: singleton FUS mutations and an unusual case with both a FUS and TARDBP mutation INTRODUCTION Mutations in the FUS gene have been shown to be a rare cause of amyotrophic lateral sclerosis (ALS-FUS) and whilst well documented clinically and genetically there have been relatively few neuropathological studies. Recent work suggested a possible correlation between pathological features such as frequency of basophilic inclusions in neurons and rate of clinical decline, other studies have revealed a discrepancy between the upper motor neuron features detected clinically and the associated pathology. The purpose of this study was to describe the pathological features associated with more recently discovered FUS mutations and reinvestigate those with well recognised mutations in an attempt to correlate the pathology with mutation and/or clinical phenotype. The brains and spinal cords of seven cases of ALS-FUS were examined neuropathologically, including cases with the newly described p. K510E mutation and a case with both a known p. P525L mutation in the FUS gene and a truncating p. Y374X mutation in the TARDBP gene. RESULTS The neuropathology in all cases revealed basophilic and FUS inclusions in the cord. The density and type of inclusions varied markedly between cases, but did not allow a clear correlation with clinical progression. Only one case showed significant motor cortical pathology despite the upper motor neuron clinical features being evident in 4 patients. The case with both a FUS and TARDBP mutation revealed FUS positive inclusions but no TDP-43 pathology. Instead there were unusual p62 positive, FUS negative neuronal and glial inclusions as well as dot-like neurites. CONCLUSIONS The study confirms cases of ALS-FUS to be mainly a lower motor neuron disease and to have pathology that does not appear to neatly correlate with clinical features or genetics. Furthermore, the case with both a FUS and TARDBP mutation reveals an intriguing pathological profile which at least in part involves a very unusual staining pattern for the ubiquitin-binding protein p62.

negative: Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate. Oscillations in patterns of expression of a large fraction of yeast genes are associated with the "metabolic cycle," usually seen only in prestarved, continuous cultures of yeast. We used FISH of mRNA in individual cells to test the hypothesis that these oscillations happen in single cells drawn from unsynchronized cultures growing exponentially in chemostats. Gene-expression data from synchronized cultures were used to predict coincident appearance of mRNAs from pairs of genes in the unsynchronized cells. Quantitative analysis of the FISH results shows that individual unsynchronized cells growing slowly because of glucose limitation or phosphate limitation show the predicted oscillations. We conclude that the yeast metabolic cycle is an intrinsic property of yeast metabolism and does not depend on either synchronization or external limitation of growth by the carbon source.

negative: A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

negative: A Tunable Diffusion‐Consumption Mechanism of Cytokine Propagation Enables Plasticity in Cell‐to‐Cell Communication in the Immune System &NA; Immune cells communicate by exchanging cytokines to achieve a context‐appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self‐assembled niches scaled with the density of cytokine‐consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80–120 &mgr;m, which resulted in a high degree of cell‐to‐cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non‐genetic cell‐to‐cell variability that is often overlooked in single‐cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors. Graphical Abstract Figure. No caption available. HighlightsCytokine penetration in tissues is governed by a diffusion‐consumption mechanismSpherical cytokine niches are generated around cytokine‐producing cellsThe characteristic niche size depends on the density of cytokine consumersCytokine niches are a source of variability in otherwise identical cells &NA; Cytokine‐mediated communication allows immune cells to achieve a context‐appropriate response, but the distance over which this communication happens is unclear. Oyler‐Yaniv et al. (2017) show that a simple diffusion‐consumption mechanism quantitatively describes the spatial spread of cytokines in vivo and results in localized niches of high cytokine concentrations that contribute to cell‐to‐cell variability.

negative: Extensive diversity of Ig-superfamily proteins in the immune system of insects. The extensive somatic diversification of immune receptors is a hallmark of higher vertebrates. However, whether molecular diversity contributes to immune protection in invertebrates is unknown. We present evidence that Drosophila immune-competent cells have the potential to express more than 18,000 isoforms of the immunoglobulin (Ig)-superfamily receptor Down syndrome cell adhesion molecule (Dscam). Secreted protein isoforms of Dscam were detected in the hemolymph, and hemocyte-specific loss of Dscam impaired the efficiency of phagocytic uptake of bacteria, possibly due to reduced bacterial binding. Importantly, the molecular diversity of Dscam transcripts generated through a mechanism of alternative splicing is highly conserved across major insect orders, suggesting an unsuspected molecular complexity of the innate immune system of insects.