The rockefeller university

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection.

Background: Magnolia champaca, commonly known as champak is a well-known tree due to its highly fragrant flowers. Champak floral scent is attributed to a complex mix of volatile organic compounds (VOCs). These aromatic flowers are widely used in flavors and fragrances industry. Despite its commercial importance, the VOC biosynthesis pathways in these flowers are largely unknown. Here, we combine metabolite and RNA sequencing (RNA-seq) analyses of fully opened champak flowers to discover the active VOC biosynthesis pathways as well as floral scent-related genes. Results: Volatile collection by headspace method and analysis by gas chromatography-mass spectrometry (GC-MS) identified a total of 43 VOCs from fully opened champak flowers, of which 46.9% were terpenoids, 38.9% were volatile esters and 5.2% belonged to phenylpropanoids/benzenoids. Sequencing and de novo assembly of champak flower transcriptome yielded 47,688 non-redundant unigenes. Transcriptome assembly was validated using standard polymerase chain reaction (PCR) based approach for randomly selected unigenes. The detailed profiles of VOCs led to the discovery of pathways and genes involved in floral scent biosynthesis from RNA-seq data. Analysis of expression levels of many floral-scent biosynthesis-related unigenes in flowers and leaves showed that most of them were expressed higher in flowers than in leaf tissues. Moreover, our metabolite-guided transcriptomics, in vitro and in vivo enzyme assays and transgenic studies identified (R)-linalool synthase that is essential for the production of major VOCs of champak flowers, (R)-linalool and linalool oxides. Conclusion: As our study is the first report on transcriptome analysis of Magnolia champaca, this transcriptome dataset that serves as an important public information for functional genomics will not only facilitate better understanding of ecological functions of champak floral VOCs, but also provide biotechnological targets for sustainable production of champak floral scent.

Mendelian susceptibility to mycobacterial disease is a rare syndrome characterized by severe clinical infections usually caused by weakly virulent mycobacterial species such as Bacillus Calmette-Guerin vaccines and environmental nontuberculous mycobacteria or more virulent mycobacteria as mycobacterium tuberculosis. Since 1996, 9 genes including 7 autosomal (STAT1, IFNGR1, IFNGR2, IL12B, IL12RB1, ISG15, and IRF8) and 2 X-linked genes ( NEMO and CYBB) have been identified. Allelic heterogeneity leaded to recognize about 18 genetic diseases with variable clinical phenotypes, but sharing a same physiological mechanism represented by a defect in human IL-12-dependant-INF-gamma-mediated immunity. We report here a case of multifocal Bacillus Calmette-Guerin osteomyelitis in a context Mendelian susceptibility to mycobacterial disease mimicking a metastatic neuroblastoma in a child presenting with delayed growth. The investigation of her twin sister showed the same disease. A heterozygous mutation in exon 22 of STAT1 gene was found in both sisters, another sister and the father being healthy and heterozygous for the same mutation.

Organizing data about patterning and morphogenesis into a coherent framework remains a challenge in developmental biology. Reporting in Science, Corson et al. (2017) apply innovative analysis to an old problem of bristle patterns in Drosophila, reducing the nonlinear interactions among tens of cells to a succinct model with quantitative predictions.

The plant immune response is a complex process involving transcriptional and posttranscriptional regulation of gene expression. Responses to plant immunity are initiated upon the perception of pathogen-associated molecular patterns, including peptide fragment of bacterial flagellin (flg22) or translation elongation factor Tu (elf18). Here, we identify an Arabidopsis thaliana long-noncoding RNA, designated ELF18-INDUCED LONG-NONCODING RNA1 (ELENA1), as a factor enhancing resistance against Pseudomonas syringe pv tomato DC3000. ELENA1 knockdown plants show decreased expression of PATHOGENESIS-RELATED GENE1 (PR1) and the plants are susceptible to pathogens. By contrast, plants overexpressing ELENA1 show elevated PR1 expression after elf18 treatment and display a pathogen resistance phenotype. RNA-sequencing analysis of ELENA1-overexpressing plants after elf18 treatment confirms increased expression of defense-related genes compared with the wild type. ELENA1 directly interacts with Mediator subunit 19a (MED19a) and affects enrichment of MED19a on the PR1 promoter. These results show that MED19a regulates PR1 expression through ELENA1. Our findings uncover an additional layer of complexity, implicating long-noncoding RNAs in the transcriptional regulation of plant innate immunity.

Mutations in the neuron-specific alpha(3) isoform of the Na+/K+-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (alpha(+/D801Y)(3)), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron in vivo recordings in awake alpha(+/D801Y)(3) mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na+/K+ exchange, but allowed the pumps to bind Na+ and become phosphorylated. These findings implicate aberrant cerebellar activity in alpha(3) isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the alpha(3) isoform Na+/K+-ATPase.

Tissue stem cells contribute to tissue regeneration and wound repair through cellular programs that can be hijacked by cancer cells. Here, we investigate such a phenomenon in skin, where during homeostasis, stem cells of the epidermis and hair follicle fuel their respective tissues. We find that breakdown of stem cell lineage confinement-granting privileges associated with both fates-is not only hallmark but also functional in cancer development. We show that lineage plasticity is critical in wound repair, where it operates transiently to redirect fates. Investigating mechanism, we discover that irrespective of cellular origin, lineage infidelity occurs in wounding when stress-responsive enhancers become activated and override homeostatic enhancers that govern lineage specificity. In cancer, stress-responsive transcription factor levels rise, causing lineage commanders to reach excess. When lineage and stress factors collaborate, they activate oncogenic enhancers that distinguish cancers from wounds.

Understanding the biologic role of N-6-methyladenosine (m(6)A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m(6)A in exons but very rarely in introns. The m(6)A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m(6)A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only similar to 10% of m(6)As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m(6)A in wild-type mouse stem cells is spliced the same in cells lacking the major m(6)A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m(6)As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m(6)A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m(6)A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval(1), juvenile interactions(2), opposite and same sex interactions(3,4,5), and territorial interactions(6). For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders(7,8,9), and more recently to understand mechanisms and evolution of vocal communication among vertebrates(10). Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds(11). The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal's preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.