Functional Crosstalk between the PP2A and SUMO Pathways Revealed by Analysis of STUbL Suppressor, razor 1-1.

Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors.

Structure and variation of CRISPR and CRISPR-flanking regions in deleted-direct repeat region Mycobacterium tuberculosis complex strains.

BACKGROUND: CRISPR and CRISPR-flanking genomic regions are important for molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) strains, and potentially for adaptive immunity to phage and plasmid DNA, and endogenous roles in the bacterium. Genotyping in the Israel National Mycobacterium Reference Center Tel-Aviv of over 1500 MTBC strains from 2008-2013 showed three strains with validated negative 43-spacer spoligotypes, that is, with putatively deleted direct repeat regions (deleted-DR/CRISPR regions). Two isolates of each of three negative spoligotype MTBC (a total of 6 isolates) were subjected to Next Generation Sequencing (NGS). As positive controls, NGS was performed for three intact-DR isolates belonging to T3_Eth, the largest multiple-drug-resistant (MDR)-containing African-origin cluster in Israel. Other controls consisted of NGS reads and complete whole genome sequences from GenBank for 20 intact-DR MTBC and for 1 deleted-DR MTBC strain recognized as CAS by its defining RD deletion. RESULTS: NGS reads from negative spoligotype MTBC mapped to reference H37Rv NC_000962.3 suggested that the DR/CRISPR regions were completely deleted except for retention of the middle IS6110 mobile element. Clonally specific deletion of CRISPR-flanking genes also was observed, including deletion of at least cas2 and cas1 genes. Genomic RD deletions defined lineages corresponding to the major spoligotype families Beijing, EAI, and Haarlem, consistent with 24 loci MIRU-VNTR profiles. Analysis of NGS reads, and analysis of contigs obtained by manual PCR confirmed that all 43 gold standard DR/CRISPR spacers were missing in the deleted-DR genomes. CONCLUSIONS: Although many negative spoligotype strains are recorded as spoligotype-international-type (SIT) 2669 in the SITVIT international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least 4 different 24 loci MIRU-VNTR and RD deletion families. We report for the first time negative spoligotype-associated total loss of CRISPR region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least CRISPR-associated genes cas2 and cas1. Since cas1 deleted E.coli shows increased sensitivity to DNA damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-DR strains and Beijing family strains as both lack the cas1 gene.

IP3 3-kinase B controls hematopoietic stem cell homeostasis and prevents lethal hematopoietic failure in mice.

Tight regulation of hematopoietic stem cell (HSC) homeostasis ensures lifelong hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify Inositol-trisphosphate 3-kinase B (Itpkb) as an essential regulator of HSC homeostasis. Young Itpkb(-/-) mice accumulated phenotypic HSC, which were less quiescent and proliferated more than wild-type (WT) controls. Itpkb(-/-) HSC downregulated quiescence and stemness associated, but upregulated activation, oxidative metabolism, protein synthesis, and lineage associated messenger RNAs. Although they had normal-to-elevated viability and no significant homing defects, Itpkb(-/-) HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb(-/-) mice lost hematopoietic stem and progenitor cells and died with severe anemia. WT HSC normally repopulated Itpkb(-/-) hosts, indicating an HSC-intrinsic Itpkb requirement. Itpkb(-/-) HSC showed reduced colony-forming activity and increased stem-cell-factor activation of the phosphoinositide-3-kinase (PI3K) effectors Akt/mammalian/mechanistic target of rapamycin (mTOR). This was reversed by treatment with the Itpkb product and PI3K/Akt antagonist IP4. Transcriptome changes and biochemistry support mTOR hyperactivity in Itpkb(-/-) HSC. Treatment with the mTOR-inhibitor rapamycin reversed the excessive mTOR signaling and hyperproliferation of Itpkb(-/-) HSC without rescuing colony forming activity. Thus, we propose that Itpkb ensures HSC quiescence and function through limiting cytokine-induced PI3K/mTOR signaling and other mechanisms.

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

BACKGROUND: Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product. RESULTS: As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels. CONCLUSIONS: This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

Noncoding transcription within the Igh distal V(H) region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells.

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eµ, the start site of Iµ germ-line transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eµ-independent. YY1(-/-) pro-B cells are greatly impaired in distal V(H) gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eµ interactions. ChIP-seq shows high level YY1 binding only at Eµ, but low levels near some antisense promoters. PAIR-Eµ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V(H) genes close to the DJ(H) rearrangement which is adjacent to Eµ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V(H) genes to undergo efficient rearrangement.

Expression of glycogenes in differentiating human NT2N neurons. Downregulation of fucosyltransferase 9 leads to decreased Lewis(x) levels and impaired neurite outgrowth.

BACKGROUND: Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. METHODS: The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. RESULTS AND CONCLUSIONS: One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis(x) motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis(x) structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewis(x), as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. GENERAL SIGNIFICANCE: These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis(x) motif plays a functional role in neurite outgrowth from human neurons.

Rad3 decorates critical chromosomal domains with gammaH2A to protect genome integrity during S-Phase in fission yeast.

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (gammaH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that gammaH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

SIGLEC12, a human-specific segregating (pseudo)gene, encodes a signaling molecule expressed in prostate carcinomas.

The primate SIGLEC12 gene encodes one of the CD33-related Siglec family of signaling molecules in immune cells. We had previously reported that this gene harbors a human-specific missense mutation of the codon for an Arg residue required for sialic acid recognition. Here we show that this R122C mutation of the Siglec-XII protein is fixed in the human population, i.e. it occurred prior to the origin of modern humans. Additional mutations have since completely inactivated the SIGLEC12 gene in some but not all humans. The most common inactivating mutation with a global allele frequency of 58% is a single nucleotide frameshift that markedly shortens the open reading frame. Unlike other CD33-related Siglecs that are primarily found on immune cells, we found that Siglec-XII protein is expressed not only on some macrophages but also on various epithelial cell surfaces in humans and chimpanzees. We also found expression on certain human prostate epithelial carcinomas and carcinoma cell lines. This expression correlates with the presence of the nonframeshifted, intact SIGLEC12 allele. Although SIGLEC12 allele status did not predict prostate carcinoma incidence, restoration of expression in a prostate carcinoma cell line homozygous for the frameshift mutation induced altered regulation of several genes associated with carcinoma progression. These stably transfected Siglec-XII-expressing prostate cancer cells also showed enhanced growth in nude mice. Finally, monoclonal antibodies against the protein were internalized by Siglec-XII-expressing prostate carcinoma cells, allowing targeting of a toxin to such cells. Polymorphic expression of Siglec-XII in humans thus has implications for prostate cancer biology and therapeutics.

Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation.

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLe(X)), and related lectin ligands on effector leukocytes. Based on anti-sLe(X) antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLe(X) formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLe(X) (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLe(X) structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLe(X) on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

Localization of Smc5/6 to centromeres and telomeres requires heterochromatin and SUMO, respectively.

The Smc5/6 holocomplex executes key functions in genome maintenance that include ensuring the faithful segregation of chromosomes at mitosis and facilitating critical DNA repair pathways. Smc5/6 is essential for viability and therefore, dissecting its chromosome segregation and DNA repair roles has been challenging. We have identified distinct epigenetic and post-translational modifications that delineate roles for fission yeast Smc5/6 in centromere function, versus replication fork-associated DNA repair. We monitored Smc5/6 subnuclear and genomic localization in response to different replicative stresses, using fluorescence microscopy and chromatin immunoprecipitation (ChIP)-on-chip methods. Following hydroxyurea treatment, and during an unperturbed S phase, Smc5/6 is transiently enriched at the heterochromatic outer repeats of centromeres in an H3-K9 methylation-dependent manner. In contrast, methyl methanesulphonate treatment induces the accumulation of Smc5/6 at subtelomeres, in an Nse2 SUMO ligase-dependent, but H3-K9 methylation-independent manner. Finally, we determine that Smc5/6 loads at all genomic tDNAs, a phenomenon that requires intact consensus TFIIIC-binding sites in the tDNAs.

Regulated and aberrant glycosylation modulate cardiac electrical signaling.

Millions afflicted with Chagas disease and other disorders of aberrant glycosylation suffer symptoms consistent with altered electrical signaling such as arrhythmias, decreased neuronal conduction velocity, and hyporeflexia. Cardiac, neuronal, and muscle electrical signaling is controlled and modulated by changes in voltage-gated ion channel activity that occur through physiological and pathological processes such as development, epilepsy, and cardiomyopathy. Glycans attached to ion channels alter channel activity through isoform-specific mechanisms. Here we show that regulated and aberrant glycosylation modulate cardiac ion channel activity and electrical signaling through a cell-specific mechanism. Data show that nearly half of 239 glycosylation-associated genes (glycogenes) were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes. The N-glycan structures produced among cardiomyocyte types were markedly variable. Thus, the cardiac glycome, defined as the complete set of glycan structures produced in the heart, is remodeled. One glycogene, ST8sia2, a polysialyltransferase, is expressed only in the neonatal atrium. Cardiomyocyte electrical signaling was compared in control and ST8sia2((-/-)) neonatal atrial and ventricular myocytes. Action potential waveforms and gating of less sialylated voltage-gated Na+ channels were altered consistently in ST8sia2((-/-)) atrial myocytes. ST8sia2 expression had no effect on ventricular myocyte excitability. Thus, the regulated (between atrium and ventricle) and aberrant (knockout in the neonatal atrium) expression of a single glycogene was sufficient to modulate cardiomyocyte excitability. A mechanism is described by which cardiac function is controlled and modulated through physiological and pathological processes that involve regulated and aberrant glycosylation.

Type 2 diabetes in mice induces hepatic overexpression of sulfatase 2, a novel factor that suppresses uptake of remnant lipoproteins.

UNLABELLED: Type 2 diabetes mellitus (T2DM) impairs hepatic clearance of atherogenic postprandial remnant lipoproteins. Our work and that of others have identified syndecan-1 heparan sulfate proteoglycans (HSPGs) as remnant lipoprotein receptors. Nevertheless, defects in the T2DM liver have not been molecularly characterized, and neither has the correction that occurs upon caloric restriction. We used microarrays to compare expression of proteoglycan-related genes in livers from control db/m mice; obese, T2DM db/db littermates fed ad libitum (AL); and db/db mice pair-fed to match the intake of db/m mice. Surprisingly, the arrays identified only one gene whose dysregulation by T2DM would disrupt HSPG structure: the heparan sulfate glucosamine-6-O-endosulfatase-2 (Sulf2). SULF2 degrades HSPGs by removing 6-O sulfate groups, but had no previously known role in diabetes or lipoprotein biology. Follow-up quantitative polymerase chain reaction assays revealed a striking 11-fold induction of Sulf2 messenger RNA in the livers of AL T2DM mice compared with controls. Immunoblots demonstrated induction of SULF2 in AL livers, with restoration toward normal in livers from pair-fed db/db mice. Knockdown of SULF2 in cultured hepatocytes doubled HSPG-mediated catabolism of model remnant lipoproteins. Notably, co-immunoprecipitations revealed a persistent physical association of SULF2 with syndecan-1. To identify mechanisms of SULF2 dysregulation in T2DM, we found that advanced glycosylation end products provoked a 10-fold induction in SULF2 expression by cultured hepatocytes and an approximately 50% impairment in their catabolism of remnants and very low-density lipoprotein, an effect that was entirely reversed by SULF2 knockdown. Adiponectin and insulin each suppressed SULF2 protein in cultured liver cells and in murine livers in vivo, consistent with a role in energy flux. Likewise, both hormones enhanced remnant lipoprotein catabolism in vitro. CONCLUSION: SULF2 is an unexpected suppressor of atherogenic lipoprotein clearance by hepatocytes and an attractive target for inhibition.

Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

The epithelial-to-mesenchymal transition (EMT) is a basic cellular process that plays a key role in normal embryonic development and in cancer progression/metastasis. Our previous study indicated that EMT processes of mouse and human epithelial cells induced by TGF-ß display clear reduction of gangliotetraosylceramide (Gg4) and ganglioside GM2, suggesting a close association of glycosphingolipids (GSLs) with EMT. In the present study, using normal murine mammary gland (NMuMG) cells, we found that levels of Gg4 and of mRNA for the UDP-Gal:ß1-3galactosyltransferase-4 (ß3GalT4) gene, responsible for reduction of Gg4, were reduced in EMT induced by hypoxia (∼1% O(2)) or CoCl(2) (hypoxia mimic), similarly to that for TGF-ß-induced EMT. An increase in the Gg4 level by its exogenous addition or by transfection of the ß3GalT4 gene inhibited the hypoxia-induced or TGF-ß-induced EMT process, including changes in epithelial cell morphology, enhanced motility, and associated changes in epithelial vs. mesenchymal molecules. We also found that Gg4 is closely associated with E-cadherin and ß-catenin. These results suggest that the ß3GalT4 gene, responsible for Gg4 expression, is down-regulated in EMT; and Gg4 has a regulatory function in the EMT process in NMuMG cells, possibly through interaction with epithelial molecules important to maintain epithelial cell membrane organization.

Reciprocal patterns of methylation of H3K36 and H3K27 on proximal vs. distal IgVH genes are modulated by IL-7 and Pax5.

The usage of >100 functional murine Ig heavy chain V(H) genes, when rearranged to D(H)J(H) genes, generates a diverse antibody repertoire. The V(H) locus encompasses 2.5 Mb, and rearrangement of V(H) genes in the D(H)-distal half of the locus are controlled very differently from the V(H) genes in the proximal end of the locus. The rearrangement of distal but not proximal V(H) genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5, or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal V(H) rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal V(H) segments and the repressive histone modification H3K27me3 is exclusively present on proximal V(H) segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal V(H) genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the V(H) locus by differentially modulating two reciprocal histone modifications during B lymphocyte development.

Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.

Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. For 95% of these genes, significant transcript levels were detected. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate-early expression. Although a similar kinetic class has been described for other virus families, to our knowledge, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other three kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome-wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression.

Dynamic transcriptome landscape in the song nucleus HVC between juvenile and adult zebra finches.

Male juvenile zebra finches learn to sing by imitating songs of adult males early in life. The development of the song control circuit and song learning and maturation are highly intertwined processes, involving gene expression, neurogenesis, circuit formation, synaptic modification, and sensory-motor learning. To better understand the genetic and genomic mechanisms underlying these events, we used RNA-Seq to examine genome-wide transcriptomes in the song control nucleus HVC of male juvenile (45 d) and adult (100 d) zebra finches. We report that gene groups related to axon guidance, RNA processing, lipid metabolism, and mitochondrial functions show enriched expression in juvenile HVC compared to the rest of the brain. As juveniles mature into adulthood, massive gene expression changes occur. Expression of genes related to amino acid metabolism, cell cycle, and mitochondrial function is reduced, accompanied by increased and enriched expression of genes with synaptic functions, including genes related to G-protein signaling, neurotransmitter receptors, transport of small molecules, and potassium channels. Unexpectedly, a group of genes with immune system functions is also developmentally regulated, suggesting potential roles in the development and functions of HVC. These data will serve as a rich resource for investigations into the development and function of a neural circuit that controls vocal behavior.

Syndecan-Fc hybrid molecule as a potent in vitro microbicidal anti-HIV-1 agent.

In the absence of a vaccine, there is an urgent need for the development of safe and effective topical microbicides to prevent the sexual transmission of human immunodeficiency virus type 1 (HIV-1). In this study, we proposed to develop a novel class of microbicides using syndecan as the antiviral agent. Specifically, we generated a soluble syndecan-Fc hybrid molecule by fusing the ectodomain of syndecan-1 to the Fc domain of a human IgG. We then tested the syndecan-Fc hybrid molecule for various in vitro microbicidal anti-HIV-1 properties. Remarkably, the syndecan-Fc hybrid molecule possesses multiple attractive microbicidal properties: (i) it blocks HIV-1 infection of primary targets including T cells, macrophages, and dendritic cells (DC); (ii) it exhibits a broad range of antiviral activity against primary HIV-1 isolates, multidrug resistant HIV-1 isolates, HIV-2, and simian immunodeficiency virus (SIV); (iii) it prevents transmigration of HIV-1 through human primary genital epithelial cells; (iv) it prevents HIV-1 transfer from dendritic cells to CD4(+) T cells; (v) it is potent when added 2 h prior to addition of HIV-1 to target cells; (vi) it is potent at a low pH; (vii) it blocks HIV-1 infectivity when diluted in genital fluids; and (viii) it prevents herpes simplex virus infection. The heparan sulfate chains of the syndecan-Fc hybrid molecule are absolutely required for HIV-1 neutralization. Several lines of evidence suggest that the highly conserved Arg298 in the V3 region of gp120 serves as the locus for the syndecan-Fc hybrid molecule neutralization. In conclusion, this study suggests that the syndecan-Fc hybrid molecule represents the prototype of a new generation of microbicidal agents that may have promise for HIV-1 prevention.

Striking decrease in the total precursor B-cell compartment during early childhood as evidenced by flow cytometry and gene expression changes.

The number of circulating B-cells in peripheral blood plateaus between 2 and 24 months of age, and thereafter declines gradually. How this reflects the kinetics of the precursor B-cell pool in the bone marrow is of clinical interest, but has not been studied thoroughly in humans. The authors analyzed bone marrow (n = 37) from healthy children and adults (flow cytometry) searching for age-related changes in the total precursor B-cell compartment. In an age-matched cohort (n = 25) they examined age-related global gene expression changes (Affymetrix) in unsorted bone marrow with special reference to the recombination activating gene 1, RAG1. Subsequently, they searched the entire gene set for transcripts correlating to the RAG1 profile to discover other known and possibly new precursor B-cell related transcripts. Both methods disclosed a marked, transient increase of total precursor B-cells at 6-20 months, followed by a rapid decrease confined to the first 2 years. The decline thereafter was considerably slower, but continued until adulthood. The relative composition of total precursor B-cells, however, did not change significantly with age. The authors identified 54 genes that were highly correlated to the RAG1 profile (r >or= .9, p < 1 x 10(-8)). Of these 54 genes, 15 were characteristically B-lineage associated like CD19, CD79, VPREB, EBF1, and PAX5; the remaining 39 previously not described as distinctively B-lineage related. The marked, transient increase in precursor B-cells and RAG1 transcriptional activity is not reflected by a similar peak in B-cells in peripheral blood, whereas the sustained plateau concurs in time.