Animal models that recapitulate human disease are proving to be an invaluable tool in the identification of novel disease-associated genes. These models can improve our understanding of the complex genetic mechanisms involved in disease and provide a basis to guide therapeutic strategies to combat these conditions. We have identified a novel mouse model of non-syndromic sensorineural hearing loss with linkage to a region on chromosome 18. Eeyore mutant mice have early onset progressive hearing impairment and show abnormal structure of the sensory epithelium from as early as 4 weeks of age. Ultrastructural and histological analyses show irregular hair cell structure and degeneration of the sensory hair bundles in the cochlea. The identification of new genes involved in hearing is central to understanding the complex genetic pathways involved in the hearing process and the loci at which these pathways are interrupted in people with a genetic hearing loss. We therefore discuss possible candidate genes within the linkage region identified in eeyore that may underlie the deafness phenotype in these mice. Eeyore provides a new model of hereditary sensorineural deafness and will be an important tool in the search for novel deafness genes.
We report the imminent completion of a set of reference genome assemblies for 16 species of Anopheles mosquitoes. In addition to providing a generally useful resource for comparative genomic analyses, these genome sequences will greatly facilitate exploration of the capacity exhibited by some Anopheline mosquito species to serve as vectors for malaria parasites. A community analysis project will commence soon to perform a thorough comparative genomic investigation of these newly sequenced genomes. Completion of this project via the use of short next-generation sequence reads required innovation in both the bioinformatic and laboratory realms, and the resulting knowledge gained could prove useful for genome sequencing projects targeting other unconventional genomes.
comparative; assembly; vector; malaria; collaboration
Human MYO7A mutations can cause a variety of conditions involving the inner ear. These include dominant and recessive non-syndromic hearing loss and syndromic conditions such as Usher syndrome. Mouse models of deafness allow us to investigate functional pathways involved in normal and abnormal hearing processes. We present two novel mouse models with mutations in the Myo7a gene with distinct phenotypes. The mutation in Myo7aI487N/I487N ewaso is located within the head motor domain of Myo7a. Mice exhibit a profound hearing loss and manifest behaviour associated with a vestibular defect. A mutation located in the linker region between the coiled-coil and the first MyTH4 domains of the protein is responsible in Myo7aF947I/F947I dumbo. These mice show a less severe hearing loss than in Myo7aI487N/I487N ewaso; their hearing loss threshold is elevated at 4 weeks old, and progressively worsens with age. These mice show no obvious signs of vestibular dysfunction, although scanning electron microscopy reveals a mild phenotype in vestibular stereocilia bundles. The Myo7aF947I/F947I dumbo strain is therefore the first reported Myo7a mouse model without an overt vestibular phenotype; a possible model for human DFNB2 deafness. Understanding the molecular basis of these newly identified mutations will provide knowledge into the complex genetic pathways involved in the maintenance of hearing, and will provide insight into recessively inherited sensorineural hearing loss in humans.
Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here, we demonstrate that memory B cell subsets unexpectedly diverge across antibody class through the differential impact of major transcriptional regulators. Conditional genetic deletion of Tbx21 selectively blocks the formation and antigen-specific response of IgG2a memory B cells in vivo. Cell intrinsic T-bet expression regulates STAT1 expression, steady-state cell survival and IgG2a BCR transcription. In contrast, RORα was differentially expressed in IgA memory B cells with siRNA knockdown and chemical inhibition supporting its selective control in cell survival and IgA BCR transcription. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function within class-specific memory B cells.
Background: Temporomandibular joint disorders (TMDs) are chronic, often refractory, pain conditions affecting the jaw and face. Patients least likely to respond to allopathic treatment have the most marked biologic responsiveness to external stressors and concomitant psychosocial and emotional difficulties. From a shamanic healing perspective, this describes individuals who are thought to be “dispirited” and may benefit from this ancient form of spiritual healing.
Objective: To report on the long-term quantitative and qualitative outcomes relative to end-of-treatment status of a phase I study that evaluated the feasibility and efficacy of shamanic healing for people with TMDs.
Methods/Design: Participants were contacted by telephone at one, three, six, and nine months after treatment and asked to report pain and disability outcomes and qualitative feedback.
Setting: Portland, OR.
Participants: Twenty-three women aged 25 to 55 years diagnosed with TMD.
Primary Outcome Measures: Participants rated their TMD-related pain and disability (on the TMD Research Diagnostic Criteria Axis II Pain Related Disability and Psychological Status Scale) at each follow-up call and were asked to describe their condition qualitatively.
Results: Improvements in usual pain, worst pain, and functional impairment reported at end of treatment did not change during the 9 months after treatment ended (p > 0.18).
Conclusion: Shamanic healing had lasting effects on TMDs in this small cohort of women.
B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T (TFH) cells. Here, we demonstrate that isotype-switched plasma cells expressed MHCII, CD80 and CD86 and intracellular machinery required for antigen presentation. Antigen-specific plasma cells could access, process and present sufficient antigen in vivo to induce multiple TH cell functions. Importantly, antigen-primed plasma cells failed to induce interleukin 21 or Bcl-6 in naïve TH cells and actively shut down these key molecules in antigen-activated TFH cells. Mice lacking plasma cells displayed altered TFH activity, providing evidence for this negative feedback loop. Hence, antigen presentation by plasma cells defines a new layer of cognate regulation that limits the antigen-specific TFH program controlling ongoing B cell immunity.
Hearing impairment is the most common sensory impairment in humans, affecting 1∶1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade.
The hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2N538K mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2N538K/N538K) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent.
Phosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function.
Neurogenin 3 (NGN3) commits pancreatic progenitors to an islet cell fate. We have induced NGN3 expression and identified upregulation of the gene encoding the Ras-associated small molecular mass GTP-binding protein, RAB3B. RAB3B localised to the cytoplasm of human β-cells, both during the foetal period and post natally. Genes encoding alternative RAB3 proteins and RAB27A were unaltered by NGN3 expression and in human adult islets their transcripts were many fold less prevalent than those of RAB3B. The regulation of insulin exocytosis in rodent β-cells and responsiveness to incretins are reliant on Rab family members, notably Rab3a and Rab27a, but not Rab3b. Our results support an important inter-species difference in regulating insulin exocytosis where RAB3B is the most expressed isoform in human islets.
Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions.
Follicular helper T (TFH) cells are a class of helper T cells specialized in the cognate control of antigen-specific B cell immunity. Upon first contact with antigen-primed B cells, pre-germinal center effector TFH cells promote B cell clonal expansion, antibody isotype switch, plasma cell differentiation and the induction of germinal centers. In contrast, within germinal centers, TFH cells regulate the fate of antigen-specific GC B cells expressing high-affinity variant B cell receptors to promote memory B cell and long-lived plasma cell development. Recent studies unravel multiple signals controlling TFH development and functional sub-types of antigen-specific TFH cells, including memory TFH cells that accelerate memory B cell responses to antigen re-challenge in vivo.
The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.
We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1Mb. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.
Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. High resolution genome wide copy number and matching expression data from 68 primary epithelial ovarian carcinomas of various histotypes was integrated to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Regions on chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (>40% of samples). Within these regions, 703/1370 (51%) unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 30% of these differentially expressed probesets also showed a strong positive correlation (r≥0.6) between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 32 known protein coding genes showed a strong positive correlation between expression and copy number. Overall, our data validates previously known ovarian cancer genes, such as ERBB2, and also identified novel potential drivers such as MYNN, PUF60 and TPX2.
The unfolded protein response (UPR) is a stress response pathway that is driven by the increased load of unfolded proteins in the endoplasmic reticulum of highly secretory cells such as plasma cells (PCs). X box binding protein 1 (XBP1) is a transcription factor that mediates one branch of the UPR and is crucial for the development of antibody-secreting PCs. PCs represent only one class of terminally differentiated B cells, however, and little is known about the role for XBP1 in the other class: memory B cells. We have developed an XBP1fl/fl CD19+/cre conditional knockout (XBP1CD19) mouse to build upon our current understanding of the function of XBP1 in PC differentiation as well as to explore the role of XBP1 in memory cell development. Using this model, we show that XBP1CD19 mice are protected from disease in an autoantibody-mediated mouse lupus model. We also identify a novel developmental stage at which B cells express the traditional PC marker CD138 (syndecan-1) but have yet to undergo XBP1-dependent functional and morphological differentiation into antibody-secreting cells. Finally, we show that memory B cells develop normally in XBP1CD19 mice, demonstrating that XBP1-mediated functions occur independently of any memory cell lineage commitment.
Allpaths2, a method for accurately assembling small genomes with high continuity using short paired reads.
We demonstrate that genome sequences approaching finished quality can be generated from short paired reads. Using 36 base (fragment) and 26 base (jumping) reads from five microbial genomes of varied GC composition and sizes up to 40 Mb, ALLPATHS2 generated assemblies with long, accurate contigs and scaffolds. Velvet and EULER-SR were less accurate. For example, for Escherichia coli, the fraction of 10-kb stretches that were perfect was 99.8% (ALLPATHS2), 68.7% (Velvet), and 42.1% (EULER-SR).
How follicular helper T cells (TFH cells) differentiate to regulate B cell immunity is critical for effective protein vaccination. Here we define three transcription factor T-bet-expressing antigen-specific effector helper T cell subsets with distinguishable function, migratory properties and developmental programming in vivo. Expression of the transcriptional repressor Blimp-1 distinguished T zone ‘lymphoid’ effector helper T cells (CD62LhiCCR7hi) from CXCR5lo ‘emigrant’ effector helper T cells and CXCR5hi ‘resident’ TFH cells expressing the transcriptional repressor Bcl-6 (CD62LloCCR7lo). We then show by adoptive transfer and intact polyclonal responses that helper T cells with the highest specific binding of peptide-major histocompatibility complex class II and the most restricted T cell antigen receptor junctional diversity ‘preferentially’ developed into the antigen-specific effector TFH compartment. Our studies demonstrate a central function for differences in the binding strength of the T cell antigen receptor in the antigen-specific mechanisms that ‘program’ specialized effector TFH function in vivo.
Follicular helper T (TFH) cells are the class of effector TH cells that regulates the stepwise development of antigen-specific B cell immunity in vivo. Deployment of CXCR5+ TFH cells to B cell zones of lymphoid tissues and stable cognate interactions with B cells are central to the delivery of antigen-specific TFH function. Recent advances help to unravel distinctive elements of developmental programming for TFH cells and unique effector TFH functions focused on antigen-primed B cells. Understanding the regulatory functions of TFH cells in the germinal center and the subsequent regulation of memory B cell responses to antigen recall represent the frontiers of this research area with the potential to alter fundamentally the design of future vaccines.
How TCR specificity evolves in vivo following protein vaccination is central to the development of T helper cell function. Most models of clonal selection in the T helper cell compartment favor TCR affinity-based thresholds. Here, we demonstrate that depot-forming vaccine adjuvants do not require TLR agonists to induce clonal dominance in antigen-specific T helper cell responses. However, readily dispersible adjuvants using TLR-9 and TLR-4 agonists skew TCR repertoire usage by increasing TCR selection thresholds and enhancing antigen-specific clonal expansion. In this manner, vaccine adjuvants control the local accumulation of T helper cells expressing TCR with the highest peptide MHC class II binding. Clonal composition was altered by mechanisms that blocked the local propagation of clonotypes independent of antigen dose and not as a consequence of inter-clonal competition. This capacity of adjuvants to modify antigen-specific Th cell clonal composition has fundamental implications for the design of future protein sub-unit vaccines.
Acute kidney injury (AKI), previously referred to as acute renal failure (ARF), represents a common and devastating problem in clinical medicine. Despite significant improvements in therapeutics, the mortality and morbidity associated with AKI remain high. A major reason for is the lack of early markers for AKI, and hence an unacceptable delay in initiating therapy. Fortunately, the application of innovative technologies such as functional genomics and proteomics to human and animal models of AKI has uncovered several novel biomarkers and therapeutic targets. The most promising of these are chronicled in this review. These include the identification of biomarker panels in plasma (NGAL and cystatin C) and urine (NGAL, KIM-1, IL-18, cystatin C, α1-microglobulin, Fetuin-A, Gro-α, and meprin). It is likely that the AKI panels will be useful for timing the initial insult, and assessing the duration and severity of AKI. It is also probable that the AKI panels will distinguish between the various etiologies of AKI, and predict clinical outcomes. It will be important in future studies to validate the sensitivity and specificity of these biomarker panels in clinical samples from large cohorts and from multiple clinical situations. Such studies will be markedly facilitated by the development of commercial tools for the reproducible measurement of biomarkers across different laboratories.
acute renal failure; proteomics; neutrophil gelatinase-associated lipocalin; cystatin C; interleukin 18; kidney injury molecule-1; biomarker panel
Distinguishing between the development of functional potential in antigen-specific T helper (Th) cells and the delivery of these specialized functions in vivo has been difficult to resolve. Here, we quantify the frequency of cytokine-producing cells within the primary and memory B10.BR Th cell response to pigeon cytochrome c (PCC). In vitro analysis of acquired functional potential indicated no Th1/Th2 cytokine polarity at the peak of the primary response with surprisingly little evidence for the selective preservation of interleukin (IL)-2, tumor necrosis factor (TNF)-α, IL-4, and interferon (IFN)-γ potentials into the memory compartment. However, the expression of these functional potentials appears tightly regulated in vivo. The staggered appearance of primary response cytokines directly ex vivo contrasts markedly with their rapid coordinate expression in the memory response. Frequencies of IL-2–, TNF-α–, IFN-γ–, and IL-10–expressing memory responders increased over their primary response counterparts, but were still markedly lower than revealed in vitro. IL-4–, IFN-γ–, and IL-10–expressing Th cells remained at low but stable frequencies over the first 6 d of the memory response. Analysis of T cell receptor β chain sequences of IL-4– and TNF-α–expressing PCC-specific Th cells provides evidence for early functional commitment among clonal progeny. These data indicate that the development of functional potential is a consequence of initial antigen experience, but delivery of specialized functions is differentially regulated in primary and memory immune responses.
immunological memory; cytokines; antigen-specific immunity; helper T cells; T cell receptor
The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220−CD138−) that are distinct from antibody-secreting B cells (B220+/−CD138+) and B220+CD138− memory B cells. These nonsecreting somatically mutated B220− memory responders rapidly dominate the splenic response and comprise >95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75–85%) and the bone marrow (>95%) expresses the B220− phenotype. Upon adoptive transfer, B220− memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220+ counterparts. The pattern of cellular differentiation after transfer indicates that B220− memory B cells act as stable self-replenishing intermediates that arise from B220+ memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220− compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long-term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function.
antigen-specific immunity; immunological memory; B lymphocyte memory; B lymphocyte subset; mice
Migration of antigen-activated CD4 T cells to B cell areas of lymphoid tissues is important for mounting T cell–dependent antibody responses. Here we show that CXC chemokine receptor (CXCR)5, the receptor for B lymphocyte chemoattractant (BLC), is upregulated on antigen-specific CD4 T cells in vivo when animals are immunized under conditions that promote T cell migration to follicles. In situ hybridization of secondary follicles for BLC showed high expression in mantle zones and low expression in germinal centers. When tested directly ex vivo, CXCR5hi T cells exhibited a vigorous chemotactic response to BLC. At the same time, the CXCR5hi cells showed reduced responsiveness to the T zone chemokines, Epstein-Barr virus–induced molecule 1 (EBI-1) ligand chemokine (ELC) and secondary lymphoid tissue chemokine (SLC). After adoptive transfer, CXCR5hi CD4 T cells did not migrate to follicles, indicating that additional changes may occur after immunization that help direct T cells to follicles. To further explore whether T cells could acquire an intrinsic ability to migrate to follicles, CD4−CD8− double negative (DN) T cells from MRL-lpr mice were studied. These T cells normally accumulate within follicles of MRL-lpr mice. Upon transfer to wild-type recipients, DN T cells migrated to follicle proximal regions in all secondary lymphoid tissues. Taken together, our findings indicate that reprogramming of responsiveness to constitutively expressed lymphoid tissue chemokines plays an important role in T cell migration to the B cell compartment of lymphoid tissues.
chemokine; CXCR5; ELC; follicle; T lymphocyte
Antigen (Ag)-driven selection of helper T cells (Th) in normal animals has been difficult to study and remains poorly understood. Using the major histocompatibility complex class II– restricted murine response to pigeon cytochrome c (PCC), we provide evidence for both preimmune and Ag-driven selection in the evolution of Ag-specific immunity in vivo. Before antigenic challenge, most Vα11+Vβ3+ Th (70%) express a critical complementarity-determining region 3 (CDR3) residue (glutamic acid at TCR-α93) associated with PCC peptide contact. Over the first 5 d of the primary response, PCC-responsive Vα11+Vβ3+ Th expressing eight preferred CDR3 features are rapidly selected in vivo. Clonal dominance is further propagated through selective expansion of the PCC-specific cells with T cell receptor (TCR) of the “best fit.” Ag-driven selection is complete before significant emergence of the germinal center reaction. These data argue that thymic selection shapes TCR-α V region bias in the preimmune repertoire; however, Ag itself and the nongerminal center microenvironment drive the selective expansion of clones with preferred TCR that dominate the response to Ag in vivo.
immunologic memory; clonal maturation; antigen-specific immunity; helper T cells; T cell receptor
Chromodomain, helicase, DNA binding 5 (CHD5) is a member of a subclass of the chromatin remodeling Swi/Snf proteins and has recently been proposed as a tumor suppressor in a diverse range of human cancers. We analyzed all 41 coding exons of CHD5 for somatic mutations in 123 primary ovarian cancers as well as 60 primary breast cancers using high-resolution melt analysis. We also examined methylation of the CHD5 promoter in 48 ovarian cancer samples by methylation-specific single-stranded conformation polymorphism and bisulfite sequencing. In contrast to previous studies, no mutations were identified in the breast cancers, but somatic heterozygous missense mutations were identified in 3 of 123 ovarian cancers. We identified promoter methylation in 3 of 45 samples with normal CHD5 and in 2 of 3 samples with CHD5 mutation, suggesting these tumors may have biallelic inactivation of CHD5. Hemizygous copy number loss at CHD5 occurred in 6 of 85 samples as assessed by single nucleotide polymorphism array. Tumors with CHD5 mutation or methylation were more likely to have mutation of KRAS or BRAF (P = .04). The aggregate frequency of CHD5 haploinsufficiency or inactivation is 16.2% in ovarian cancer. Thus, CHD5 may play a role as a tumor suppressor gene in ovarian cancer; however, it is likely that there is another target of the frequent copy number neutral loss of heterozygosity observed at 1p36.