Search

Axon outgrowth is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. We find that mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal outgrowth, yet have little or no defect in neuronal differentiation or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-1 with efficient axonal outgrowth. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these embryonic axons to respond to growth factors with rapid outgrowth requires activation of calcineurin/NFAT signaling by these factors. The precise parsing of signals for elongation turning and survival could allow independent control of these processes during development.

cope The uptake of dietary plant small RNAs (sRNAs) in consumers remains controversial, which is mainly due to low dietary content in combination with poor fractional absorption. MIR2911, among all the plant sRNAs including microRNAs, has been shown to be one of the most robustly absorbed sRNAs. Here we analyze the unusual abundance and unique genesis of MIR2911 during vegetable processing. Methods and results Using qRT-PCR, the abundance of MIR2911 increased dramatically in macerated tissues while other microRNAs degraded. The accumulation of MIR2911 correlated with the degradation of the rRNAs, consistent with MIR2911 being derived from the 26S rRNA. Bioinformatic analysis predicts a microRNA-like precursor structure for MIR2911; however, no reciprocal increase in the putative star-strand was noted, and using an Arabidopsis mutation deficient in miRNA processing the accumulation of MIR2911 appeared Dicer independent. MIR2911 was incorporated into the mammalian RNA-induced silencing complex as demonstrated in HEK293T cells, where transfected synthetic MIR2911 modestly suppressed the activity of a cognate luciferase reporter. Conclusion The genesis and amplification of MIR2911 post-harvest is atypical, as traditional plant bioactives are less plentiful as vegetables lose freshness. These findings offer an explanation to the disparity in serum detection between MIR2911 and canonical plant-based miRNAs.

The importance of translational regulation in tumorigenesis and metastasis is increasingly appreciated. We have leveraged polyribosomal profiling to prospectively and functionally define translational regulatory programs underlying the epithelial to mesenchymal transition (EMT), one of the early manifestations of metastasis, in breast epithelial cells. Our approach identified scores of mRNAs both enriched and depleted from polyribosomal fractions in mesenchymal cells as compared to their epithelial counterparts. Computational analysis of the 3' untranslated regions (UTRs) of mRNAs enriched in polysomal fractions derived from the mesenchymal state revealed enrichment of a distinct GU-rich cis-element within these mRNA transcripts. Strikingly, while relative representation of these GU-rich-containing transcripts at the level of bulk mRNA were generally decreased in mesenchymal cells, the protein encoded by each of these mRNAs was dramatically increased. 3' UTRs from affected transcripts conferred similar regulation upon a fluorescent reporter gene dependent upon the presence of the GU-rich element within the 3' UTR sequence. Several of the translationally upregulated transcripts were necessary and/or sufficient for the EMT program. We next identified the CELF1 protein as a common regulator of these mRNA transcripts, demonstrating direct binding of the CELF1 protein to affected transcripts dependent upon the presence of the GU-rich element within their 3' UTRs. CELF1 was itself necessary and sufficient for EMT in several distinct in vitro models as well as for metastatic colonization in vivo. Analysis of publicly available transcriptomic data revealed no increase in the relative expression of CELF1's regulatory targets or CELF1 itself in human breast cancer. We demonstrate that the CELF1 gene product is itself post-translationally regulated during EMT, and that CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues and correlates with disease progression. Cumulatively, our data present an eleven component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumor metastasis in human breast cancer. Citation Format: Arindam Chaudhury, Shebna Cheema, Joseph M. Fachini, Natee Kongchan, Guojun Lu, Lukas M. Simon, Tao Wang, Sufeng Mao, Daniel G. Rosen, Michael M. Ittmann, Susan G. Hilsenbeck, Chad A. Shaw, Joel R. Neilson. CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A34.

The purpose of this study is to understand translational mechanisms underlying the cooperativity between FGF and WNT signaling in cancer progression. FGF and WNT signaling, crucial pathways in mammary stem cell self-renewal, are frequently deregulated in human cancer. We previously showed that acute activation of FGF signaling in the mammary gland of pubertal mice with WNT hyperactivation dramatically accelerates cancer progression. FGF-WNT-hyperactive tumor cells exhibit enhanced activation of translational machinery. Given the potential involvement of translational regulation in FGF-WNT induced tumorigenesis, we employed ribosome profiling to identify selectively translated and ribosome associated RNAs induced by acute FGF activation in vivo. Many genes associated with RNA processing and transport were translationally upregulated. Interestingly, the most translationally upregulated transcript was Malat1, originally thought to be a nuclear-restricted long noncoding RNA (lncRNA) that regulates transcription of cancer-associated genes. Along with Malat1, many lncRNAs were associated with ribosomes in FGF-WNT tumors. Malat1's association with polysomes upon acute FGF activation was confirmed by direct polysome analysis. We also demonstrated that Malat1 transcripts are transported to the cytoplasm during G2/M transition, implicating that Malat1 may be translated into small novel peptides or involved in translation regulation during this particular phase of cell cycle. Additionally, there is evidence that Malat1 is a direct downstream target of WNT signaling. Our findings indicate that FGF activation potentially increases the translation efficiency of WNT-target oncogenes including lncRNAs like Malat1, unraveling a novel mechanism of crosstalk between lncRNAs and translational regulation in cancer progression. Citation Format: Tuan M. Nguyen, Elena B. Kabotyanski, Lucas C. Reineke, Qianxing Mo, Peng Zhang, Xiang Zhang, Yiwen Chen, Joel Neilson, Richard E. Lloyd, Jeffrey M. Rosen. FGF-WNT cooperativity and the role of lncRNAs in translational regulation in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A03.

MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs. Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts. While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly. Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.

If human immunodeficiency virus type 1 (HIV-1) vaccines are to be highly effective, it is essential to understand the virologic factors that contribute to HIV-1 transmission. It is likely that transmission is determined, in part, by the genotype or phenotype (or both) of infectious virus present in the index case, which in turn will influence the quantity of virus that may be exchanged during sexual contact. Transmission may also depend on the fitness of the virus for replication in the exposed individual, which may be influenced by whether a virus encounters a target cell that is susceptible to infection by that specific variant. Of interest, our data suggest that the complexity of the virus that is transmitted may be different in female and male sexual exposures.

In sub-Saharan Africa, where the effects of human immunodeficiency virus type 1 (HIV-1) have been most devastating, there are multiple subtypes of this virus. The distribution of different subtypes within African populations is generally not linked to particular risk behaviors. Thus, Africa is an ideal setting in which to examine the diversity and mixing of viruses from different subtypes on a population basis. In this setting, it is also possible to address whether infection with a particular subtype is associated with differences in disease stage. To address these questions, we analyzed the HIV-1 subtype, plasma viral loads, and CD4 lymphocyte levels in 320 women from Nairobi, Kenya. Subtype was determined by a combination of heteroduplex mobility assays and sequence analyses of envelope genes, using geographically diverse subtype reference sequences as well as envelope sequences of known subtype from Kenya. The distribution of subtypes in this population was as follows: subtype A, 225 (70.3%); subtype D, 65 (20.5%); subtype C, 22 (6.9%); and subtype G, 1 (0.3%). Intersubtype recombinant envelope genes were detected in 2.2% of the sequences analyzed. Given that the sequences analyzed represented only a small fraction of the proviral genome, this suggests that intersubtype recombinant viral genomes may be very common in Kenya and in other parts of Africa where there are multiple subtypes. The plasma viral RNA levels were highest in women infected with subtype C virus, and women infected with subtype C virus had significantly lower CD4 lymphocyte levels than women infected with the other subtypes. Together, these data suggest that women in Kenya who are infected with subtype C viruses are at more advanced stages of immunosuppression than women infected with subtype A or D. There are at least two models to explain the data from this cross-sectional study; one is that infection with subtype C is associated with a more rapid disease progression, and the second is that subtype C represents an older epidemic in Kenya. Discriminating between these possibilities in a longitudinal study will be important for increasing our understanding of the role of specific subtypes in the transmission and pathogenesis of HIV-1.

Validation and comparison of previously unknown microRNA genes in related herpesviruses yielded several surprises, most notably in regard to viral evolution and microRNA biogenesis. An explosion of literature has recently appeared describing the identification and mechanism of action of microRNAs (miRNAs)—small RNA regulators of gene expression in plant and animal cells.

Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1.

The importance of translational regulation in tumorigenesis and metastasis is increasingly appreciated. We have leveraged polyribosomal profiling to prospectively and functionally define translational regulatory programs underlying the epithelial to mesenchymal transition (EMT), one of the early manifestations of metastasis, in breast epithelial cells. Our approach identified scores of mRNAs both enriched and depleted from polyribosomal fractions in mesenchymal cells as compared to their epithelial counterparts. Computational analysis of the 3′ untranslated regions (UTRs) of mRNAs enriched in polysomal fractions derived from the mesenchymal state revealed enrichment of a distinct GU-rich cis-element within these mRNA transcripts. Strikingly, while relative representation of these GU-rich-containing transcripts at the level of bulk mRNA were generally decreased in mesenchymal cells, the protein encoded by each of these mRNAs was dramatically increased. 3′ UTRs from affected transcripts conferred similar regulation upon a fluorescent reporter gene dependent upon the presence of the GU-rich element within the 3′ UTR sequence. Several of the translationally upregulated transcripts were necessary and/or sufficient for the EMT program. We next identified the CELF1 protein as a common regulator of these mRNA transcripts, demonstrating direct binding of the CELF1 protein to affected transcripts dependent upon the presence of the GU-rich element within their 3′ UTRs. CELF1 was itself necessary and sufficient for EMT in several distinct in vitro models as well as for metastatic colonization in vivo. Analysis of publicly available transcriptomic data revealed no increase in the relative expression of CELF1's regulatory targets or CELF1 itself in human breast cancer. We demonstrate that the CELF1 gene product is itself post-translationally regulated during EMT, and that CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues and correlates with disease progression. Cumulatively, our data present a distinctive model of gene regulation, invisible to traditional transcriptional profiling, in which the CELF1 protein serves as a central node controlling translational activation of the EMT program and ultimately tumor metastasis in human breast cancer. Citation Format: Arindam Chaudhury, Natee Kongchan, Joseph M. Fachini, Lukas M. Simon, Tao Wang, Sufeng Mao, Daniel G. Rosen, Michael M. Ittmann, Susan G. Hilsenbeck, Chad A. Shaw, Joel R. Neilson. CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B58.

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.

Next generation sequencing (NGS) is increasingly being used for transcriptome-wide analysis of differential gene expression. The NGS data are multidimensional count data. Therefore, most of the statistical methods developed well for microarray data analysis are not applicable to transcriptomic data. For this reason, a variety of new statistical methods based on count data of transcript reads have been correspondingly proposed. But due to high cost and limitation of biological resources, current NGS data are still generated from a few replicate libraries. Some of these existing methods do not always have desirable performances on count data. We here developed a very powerful and robust statistical method based on beta and binomial distributions. Our method (mBeta t-test) is specifically applicable to sequence count data from small samples. Both simulated and real transcriptomic data showed mBeta t-test significantly outperformed the existing top statistical methods chosen in all 12 given scenarios and performed with high efficiency and high stability. The differentially expressed genes found by our method from real transcriptomic data were validated by qPCR experiments. Our method shows high power in finding truly differential expression, conservatively estimating FDR and high stability in RNA sequence count data derived from small samples. Our method can also be extended to genome-wide detection of differential splicing events.

The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin Abeta gene-targeted mice, which show a approximately 50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin Abeta-/- and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin Abeta-/- mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of gene-targeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overload-mediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1- or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.

During development, discrete cell fates often result from variation in the intensity of a particular signal. The mechanisms underlying these seemingly analog-to-digital switches are not understood. In developing T lymphocytes, low-intensity signals through the antigen receptor result in positive selection while more intense signals give rise to negative selection. By deleting the genetic locus encoding the regulatory B1 subunit of calcineurin specifically in thymocytes, we found an absolute requirement for calcineurin in positive selection. In contrast, calcineurin activity was dispensable in several models of negative selection. Unexpectedly, we found that removal of calcineurin activity from thymocytes results in inefficient ERK activation at the double-positive stage of thymocyte development, when selection occurs. These studies clarify the mechanism by which graded signals are converted to discrete outcomes in T cell development and further indicate that the developmental roles of calcineurin likely contribute to immunosuppression by calcineurin inhibitors.

The modulation of intracellular calcium ion concentration, [Ca(2+)](i), is a common signalling mechanism used in many biological systems. B and T lymphocytes rely on Ca(2+) signalling to initiate both developmental and activation programs. Recent data has shed new light on the initiation of this signalling pathway, the connection between the release of intracellular Ca(2+) stores and the influx of extracellular Ca(2+), and the molecular identity of the elusive Ca(2+) release-activated Ca(2+) (CRAC) channel. In addition, recent gene profiling of T lymphocytes has identified the genes that are controlled by [Ca(2+)](i) and the Ca(2+)-dependent phosphatase calcineurin.

Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3' untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3' UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3' UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome--a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3' UTR of interest. The pBUTR (piggyBac-based 3' UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.

Recent structural studies have supported a kinetic model of TCR activation, raising the question of how the duration of receptor occupancy is translated into activation of immune response genes. We summarize evidence that the cytoplasmic-to-nuclear shuttling of NF-ATc family members monitors the duration of receptor occupancy.

The molecular basis of learning and memory has been the object of several recent advances, which have focused attention on calcium-regulated pathways controlling transcription. One of the molecules implicated by pharmacological, biochemical and genetic approaches is the calcium/calmodulin-regulated phosphatase, calcineurin. In lymphocytes, calcineurin responds to specific calcium signals and regulates expression of several immediate early genes by controlling the nuclear import of the NF-ATc family of transcription factors. Here we show that NF-ATc4/NF-AT3 in hippocampal neurons can rapidly translocate from cytoplasm to nucleus and activate NF-AT-dependent transcription in response to electrical activity or potassium depolarization. The calcineurin-mediated translocation is critically dependent on calcium entry through L-type voltage-gated calcium channels. GSK-3 can phosphorylate NF-ATc4, promoting its export from the nucleus and antagonizing NF-ATc4-dependent transcription. Furthermore, we show that induction of the inositol 1,4,5-trisphosphate receptor type 1 is controlled by the calcium/calcineurin/NF-ATc pathway. This provides a new perspective on the function of calcineurin in the central nervous system and indicates that NF-AT-mediated gene expression may be involved in the induction of hippocampal synaptic plasticity and memory formation.

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific naïve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to naïve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to naïve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.

This year, the Lasker Foundation recognizes Victor Ambros, Gary Ruvkun, and David Baulcombe for their pioneering work elucidating the role of short RNA species in the posttranscriptional regulation of eukaryotic gene expression.

The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.

SummaryMicroRNAs (miRNAs) are noncoding RNAs that suppress translation of specific mRNAs. The miRNA machinery interacts with fragile X mental retardation protein (FMRP), which functions as translational repressor. We show that miR-125b and miR-132, as well as several other miRNAs, are associated with FMRP in mouse brain. miR-125b and miR-132 had largely opposing effects on dendritic spine morphology and synaptic physiology in hippocampal neurons. FMRP knockdown ameliorates the effect of miRNA overexpression on spine morphology. We identified NMDA receptor subunit NR2A as a target of miR-125b and show that NR2A mRNA is specifically associated with FMRP in brain. In hippocampal neurons, NR2A expression is negatively regulated through its 3′ UTR by FMRP, miR-125b, and Argonaute 1. Regulation of NR2A 3′UTR by FMRP depends in part on miR-125b. Because NMDA receptor subunit composition profoundly affects synaptic plasticity, these observations have implications for the pathophysiology of fragile X syndrome, in which plasticity is altered.

BACKGROUND: Global genetic diversity of HIV-1 is a major challenge to the development of HIV vaccines. We aimed to estimate the regional and global distribution of HIV-1 subtypes and recombinants during 1990-2015. METHODS: We searched PubMed, EMBASE (Ovid), CINAHL (Ebscohost), and Global Health (Ovid) for HIV-1 subtyping studies published between Jan 1, 1990, and Dec 31, 2015. We collected additional unpublished HIV-1 subtyping data through a global survey. We included prevalence studies with HIV-1 subtyping data collected during 1990-2015. We grouped countries into 14 regions and analysed data for four time periods (1990-99, 2000-04, 2005-09, and 2010-15). The distribution of HIV-1 subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs) in individual countries was weighted according to the UNAIDS estimates of the number of people living with HIV (PLHIV) in each country to generate regional and global estimates of HIV-1 diversity in each time period. The primary outcome was the number of samples designated as HIV-1 subtypes A, B, C, D, F, G, H, J, K, CRFs, and URFs. The systematic review is registered with PROSPERO, number CRD42017067164. FINDINGS: This systematic review and global survey yielded 2203 datasets with 383 519 samples from 116 countries in 1990-2015. Globally, subtype C accounted for 46·6% (16 280 897/34 921 639 of PLHIV) of all HIV-1 infections in 2010-15. Subtype B was responsible for 12·1% (4 235 299/34 921 639) of infections, followed by subtype A (10·3%; 3 587 003/34 921 639), CRF02_AG (7·7%; 2 705 110/34 921 639), CRF01_AE (5·3%; 1 840 982/34 921 639), subtype G (4·6%; 1 591 276/34 921 639), and subtype D (2·7%; 926 255/34 921 639). Subtypes F, H, J, and K combined accounted for 0·9% (311 332/34 921 639) of infections. Other CRFs accounted for 3·7% (1 309 082/34 921 639), bringing the proportion of all CRFs to 16·7% (5 844 113/34 921 639). URFs constituted 6·1% (2 134 405/34 921 639), resulting in recombinants accounting for 22·8% (7 978 517/34 921 639) of all global HIV-1 infections. The distribution of HIV-1 subtypes and recombinants changed over time in countries, regions, and globally. At a global level during 2005-15, subtype B increased, subtypes A and D were stable, and subtypes C and G and CRF02_AG decreased. CRF01_AE, other CRFs, and URFs increased, leading to a consistent increase in the global proportion of recombinants over time. INTERPRETATION: Global and regional HIV diversity is complex and evolving, and is a major challenge to HIV vaccine development. Surveillance of the global molecular epidemiology of HIV-1 remains crucial for the design, testing, and implementation of HIV vaccines. FUNDING: None. ispartof: LANCET INFECTIOUS DISEASES vol:19 issue:2 pages:143-155 ispartof: location:United States status: published