DEP-Domain-Mediated Regulation of GPCR Signaling Responses

G protein-coupled receptors (GPCRs) mediate cellular responses to a variety of stimuli, but how specific responses are regulated has been elusive, as the types of GPCRs vastly outnumber the classes of G protein heterotrimers available to initiate downstream signaling. In our analysis of signaling proteins containing DEP domains ( approximately 90 residue sequence motifs first recognized in fly Dishevelled, worm EGL-10, and mammalian Pleckstrin), we find that DEP domains are responsible for specific recognition of GPCRs. We examined the yeast regulator of G protein signaling (RGS) protein Sst2 and demonstrate that the DEP domains in Sst2 mediate binding to its cognate GPCR (Ste2). DEP-domain-mediated tethering promotes downregulation by placing the RGS protein in proximity to its substrate (receptor-activated Galpha subunit). Sst2 docks to the Ste2 cytosolic tail, but only its unphosphorylated state, allowing for release and recycling of this regulator upon receptor desensitization and internalization. DEP-domain-mediated targeting of effectors and regulators to specific GPCRs provides a means to dictate the nature, duration, and specificity of the response

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies

Dependence and Imperialism in India

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69044/2/10.1177_048661347300500111.pd

Genomic Profiling of a Randomized Trial of Interferon-α versus Hydroxyurea in MPN Reveals Mutation-Specific Responses

Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα–treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment

Sporadic hemangioblastomas are characterized by cryptic VHL inactivation

Abstract Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.http://deepblue.lib.umich.edu/bitstream/2027.42/110224/1/40478_2014_Article_167.pd

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus.BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus.Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic

Genetic and Pharmacological Inhibition of MicroRNA-92a Maintains Podocyte Cell Cycle Quiescence and Limits Crescentic Glomerulonephritis

Crescentic rapidly progressive glomerulonephritis (RPGN) represents the most aggressive form of acquired glomerular disease. While most therapeutic approaches involve potentially toxic immunosuppressive strategies, the pathophysiology remains incompletely understood. Podocytes are glomerular epithelial cells that are normally growth-arrested because of the expression of cyclin-dependent kinase (CDK) inhibitors. An exception is in RPGN where podocytes undergo a deregulation of their differentiated phenotype and proliferate. Here we demonstrate that microRNA-92a (miR-92a) is enriched in podocytes of patients and mice with RPGN. The CDK inhibitor p57Kip2 is a major target of miR-92a that constitutively safeguards podocyte cell cycle quiescence. Podocyte-specific deletion of miR-92a in mice de-repressed the expression of p57Kip2 and prevented glomerular injury in RPGN. Administration of an anti-miR-92a after disease initiation prevented albuminuria and kidney failure, indicating miR-92a inhibition as a potential therapeutic strategy for RPGN. We demonstrate that miRNA induction in epithelial cells can break glomerular tolerance to immune injury

Genome-wide association study of placental weight in 65,405 newborns and 113,620 parents reveals distinct and shared genetic influences between placental and fetal growth

A well-functioning placenta is essential for fetal and maternal health throughout pregnancy. Using placental weight as a proxy for placental growth, we report genome-wide association analyses in the fetal (n = 65,405), maternal (n = 61,228) and paternal (n = 52,392) genomes, yielding 40 independent association signals. Twenty-six signals are classified as fetal, four maternal and three fetal and maternal. A maternal parent-of-origin effect is seen near KCNQ1. Genetic correlation and colocalization analyses reveal overlap with birth weight genetics, but 12 loci are classified as predominantly or only affecting placental weight, with connections to placental development and morphology, and transport of antibodies and amino acids. Mendelian randomization analyses indicate that fetal genetically mediated higher placental weight is causally associated with preeclampsia risk and shorter gestational duration. Moreover, these analyses support the role of fetal insulin in regulating placental weight, providing a key link between fetal and placental growth