Leptin treatment ameliorates lipoatrophic diabetes in animal models and humans. Transgenic mice overexpressing leptin (LepTg) are lipoatrophic but not diabetic and thus represent a model for elucidating mechanisms of leptin-mediated glucose homeostasis. In this communication, we show that LepTg mice overexpress the forkhead transcription factor foxo4 in their remnant adipose tissue. To further characterize the role of foxo4 in adipose tissue, we generated transgenic mice overexpressing a constitutive active form of foxo4 (A3foxo4) under the control of the aP2 promoter/enhancer. aP2-A3foxo4 mice are not lipoatrophic but are able to clear glucose rapidly similar to LepTg mice. In addition, both LepTg and A3foxo4 mice show in their adipocytes increased AMP-activated protein kinase (AMPK) phosphorylation, suggesting a link between AMPK, glucose clearance, foxo4 and the leptin axis. These studies shed new light on mechanisms by which leptin treatment improves glucose disposal.

Genome-wide interaction-based association (GWIBA) analysis has the potential to identify novel susceptibility loci. These interaction effects could be missed with the prevailing approaches in genome-wide association studies (GWAS). However, no convincing loci have been discovered exclusively from GWIBA methods, and the intensive computation involved is a major barrier for application. Here, we developed a fast, multi-thread/parallel program named "pair-wise interaction-based association mapping" (PIAM) for exhaustive two-locus searches. With this program, we performed a complete GWIBA analysis on seven diseases with stringent control for false positives, and we validated the results for three of these diseases. We identified one pair-wise interaction between a previously identified locus, C1orf106, and one new locus, TEC, that was specific for Crohn's disease, with a Bonferroni corrected P < 0.05 (P = 0.039). This interaction was replicated with a pair of proxy linked loci (P = 0.013) on an independent dataset. Five other interactions had corrected P < 0.5. We identified the allelic effect of a locus close to SLC7A13 for coronary artery disease. This was replicated with a linked locus on an independent dataset (P = 1.09 × 10⁻⁷). Through a local validation analysis that evaluated association signals, rather than locus-based associations, we found that several other regions showed association/interaction signals with nominal P < 0.05. In conclusion, this study demonstrated that the GWIBA approach was successful for identifying novel loci, and the results provide new insights into the genetic architecture of common diseases. In addition, our PIAM program was capable of handling very large GWAS datasets that are likely to be produced in the future.

Results of the recently published ONTARGET study (The Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial) showed that telmisartan (80 mg/day) was non-inferior to ramipril (10 mg/day) in reducing cardiovascular events. Clinicians in Asia doubt tolerability of these doses for their patients. We therefore analyzed data from this study and a parallel study TRANSCEND (Telmisartan Randomized Assessment Study in ACE Intolerant Subjects with Cardiovascular Disease). Our objectives were to compare Asians and non-Asians with respect to the following: 1) Effectiveness of telmisartan vs. ramipril in reducing cardiovascular events;2) Proportions who reached the full dose of telmisartan, ramipril or placebo; and3) Proportions of overall discontinuations, and discontinuations due to adverse effects.

To understand the principles underlying protein folding, many molecular modeling methods are being developed for predicting functional positions. In this work, fully flexible dinucleotides d(pApA), d(pApC), d(pApG), d(pApT), d(pCpA), d(pCpC), d(pCpG), d(pCpT), d(pGpA), d(pGpC), d(pGpG), d(pGpT), d(pTpA), d(pTpC), d(pTpG), and d(pTpT) were first docked onto the surface of scorpion polypeptide toxins (LqhIT2, PDB ID:2I61) and homology modeled ANEPIII. Automated docking was able to identify sites on scorpion polypeptide toxins where favorable nucleotide interactions can occur, and those sites were in agreement with the mutation data of this protein published recently [I. Karbat, R. Kahn, L. Cohen, N. Ilan, N. Gilles, G. Corzo, O. Froy, M. Gur, G. Albrecht, S.H. Heinemann, D. Gordon, M. Gurevitz, The unique pharmacology of the scorpion alpha-like toxin Lqh3 is associated with its flexible C-tail, Febs J 274 (2007) 1918-1931]. Simulation results suggested that dinucleotides docking is a suitable molecular modeling method that could be developed for protein functional site recognition.

Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level.

The number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in synapses determines synaptic strength. AMPAR expression can be regulated locally in dendrites by synaptic activity. The mechanisms of activity-dependent local regulation of AMPAR expression, however, remain unclear. Here, we tested whether microRNAs (miRNAs) are involved in N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent AMPAR expression. We used the 3' untranslated region of Gria1, which encodes the AMPA receptor subunit GluA1, to pull down miRNAs binding to it and analyzed these miRNAs using next-generation deep sequencing. Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA. We confirmed that miR-501-3p targets Gria1 and regulates its expression under physiological conditions. The expression of miR-501-3p and GluA1, moreover, is inversely correlated during postnatal brain development. miR-501-3p expression is up-regulated locally in dendrites through the NMDAR subunit GluN2A, and this regulation is required for NMDA-induced suppression of GluA1 expression and long-lasting remodeling of dendritic spines. These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

The B-cell receptor (BCR) signaling pathway has gained significant attention as a therapeutic target in B-cell malignancies. Recently, several drugs that target the BCR signaling pathway, especially the Btk inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicates that pharmacological inhibition of BCR pathway holds promise in B-cell lymphoma treatment. Here we present a novel covalent irreversible Btk inhibitor PLS-123 with more potent anti-proliferative activity compared with ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and dual-action inhibitory mode of Btk activation. The phosphorylation of BCR downstream activating AKT/mTOR and MAPK signal pathways was also more significantly reduced after treatment with PLS-123 than ibrutinib. Gene expression profile analysis further suggested that the different selectivity profile of PLS-123 led to significant downregulation of oncogenic gene PTPN11 expression, which might also offer new opportunities beyond what ibrutinib has achieved. In addition, PLS-123 dose-dependently attenuated BCR- and chemokine-mediated lymphoma cell adhesion and migration. Taken together, Btk inhibitor PLS-123 suggested a new direction to pharmacologically modulate Btk function and develop novel therapeutic drug for B-cell lymphoma treatment.

Enrichment of glycopeptides by hydrazide chemistry (HC) is a popular method for glycoproteomics analysis. However, possible side reactions of peptide backbones during the glycan oxidation in this method have not been comprehensively studied. Here, we developed a proteomics approach to locate such side reactions and found several types of the side reactions that could seriously compromise the performance of glycoproteomics analysis. Particularly, the HC method failed to identify N-terminal Ser/Thr glycopeptides because the oxidation of vicinal amino alcohol on these peptides generates aldehyde groups and after they are covalently coupled to HC beads, these peptides cannot be released by PNGase F for identification. To overcome this drawback, we apply a peptide N-terminal protection strategy in which primary amine groups on peptides are chemically blocked via dimethyl labeling, thus the vicinal amino alcohols on peptide N-termini are eliminated. Our results showed that this strategy successfully prevented the oxidation of peptide N-termini and significantly improved the coverage of glycoproteome.

Retinogenesis is a precisely controlled developmental process during which different types of neurons and glial cells are generated under the influence of intrinsic and extrinsic factors. Three transcription factors, Foxn4, RORβ1 and their downstream effector Ptf1a, have been shown to be indispensable intrinsic regulators for the differentiation of amacrine and horizontal cells. At present, however, it is unclear how Ptf1a specifies these two cell fates from competent retinal precursors. Here, through combined bioinformatic, molecular and genetic approaches in mouse retinas, we identify the Tfap2a and Tfap2b transcription factors as two major downstream effectors of Ptf1a. RNA-seq and immunolabeling analyses show that the expression of Tfap2a and 2b transcripts and proteins is dramatically downregulated in the Ptf1a null mutant retina. Their overexpression is capable of promoting the differentiation of glycinergic and GABAergic amacrine cells at the expense of photoreceptors much as misexpressed Ptf1a is, whereas their simultaneous knockdown has the opposite effect. Given the demonstrated requirement for Tfap2a and 2b in horizontal cell differentiation, our study thus defines a Foxn4/RORβ1-Ptf1a-Tfap2a/2b transcriptional regulatory cascade that underlies the competence, specification and differentiation of amacrine and horizontal cells during retinal development.

Hepatocellular carcinoma (HCC) is a malignant tumor. The morbidity and mortality of HCC tend to ascend and become a serious threat to the population health. Genetic studies of HCC have identified several susceptibility loci of HCC. In this study, we aim to replicate the association of these loci in our samples from Chinese population and further investigate the genetic interaction. We selected 16 SNPs within 1p36.22, 2q32.2-q32.3, 3p21.33, 8p12, 14q32.11 and 21q21.3 and genotyped in 507 HCC patients and 3014 controls by using Sequenom MassARRAY system. Association analyses were performed by using PLINK 1.07. We observed that the STAT4 (2q32.2-q32.3) at rs7574865 (P=1.17×10(-3), OR=0.79) and EFCAB11 (14q32.11) at rs8013403 (P=1.54×10(-3), OR=0.80) were significantly associated with HCC in this study. In 3p21.33, genetic variant rs6795737 within GLB1 was also observed with suggestive evidence (P=9.98×10(-3), OR=0.84). In the interaction analysis, the pair of associated SNPs (rs7574865 within STAT4, rs8013403 within EFCAB11) generated evidence for interaction (P=4.10×10(-3)). In summary, our work first reported the association of 14q32.11 (EFCAB11) with HCC in Chinese Han population and revealed the genetic interaction between STAT4 (2q32.2-q32.3) and EFCAB11 (14q32.11) in HCC.

The analysis of individual gene product should enable to clarify the role of a particular enzyme in a complex xylanase system of A. niger. The two genes encoding precursors of co-produced endo-1,4-β-D-xylanases, xynA1 and xynB, were isolated from Aspergillus niger SCTCC 400264 (SCTCC, China) by using RT-PCR technique and then successfully expressed in Escherichia coli BL21. The nucleotide sequences of the xynA1 and xynB genes revealed that they were only 52.5% homology to each other. Characterization of the recombinant enzymes revealed the different properties: the specific activity of recombinant XYNA1 was 16.58 U/mg compared to 1201.7 U/mg for recombinant XYNB; The optimum temperature and pH of the recombinant XYNA1 were 35 °C and 3.0, respectively, whereas the corresponding values for the recombinant XYNB were 55 °C and 5.0, respectively; The recombinant XYNB showed much more thermostability than recombinant XYNA1; The recombinant XYNB showed 94% of maximal activity after incubating in water for 60 min at 60 °C compared to no activity for recombinant XYNA1. Various metal ions had different effects on activity between the two recombinant xylanases.

Using expression profiles from postmortem prefrontal cortex samples of 624 dementia patients and non-demented controls, we investigated global disruptions in the co-regulation of genes in two neurodegenerative diseases, late-onset Alzheimer's disease (AD) and Huntington's disease (HD). We identified networks of differentially co-expressed (DC) gene pairs that either gained or lost correlation in disease cases relative to the control group, with the former dominant for both AD and HD and both patterns replicating in independent human cohorts of AD and aging. When aligning networks of DC patterns and physical interactions, we identified a 242-gene subnetwork enriched for independent AD/HD signatures. This subnetwork revealed a surprising dichotomy of gained/lost correlations among two inter-connected processes, chromatin organization and neural differentiation, and included DNA methyltransferases, DNMT1 and DNMT3A, of which we predicted the former but not latter as a key regulator. To validate the inter-connection of these two processes and our key regulator prediction, we generated two brain-specific knockout (KO) mice and show that Dnmt1 KO signature significantly overlaps with the subnetwork (P = 3.1 × 10(-12)), while Dnmt3a KO signature does not (P = 0.017).

The SWI/SNF complex plays an important role in mouse embryonic stem cells (mESCs), but it remains to be determined whether this complex is required for the pluripotency of human ESCs (hESCs). Using RNAi, we demonstrated that depletion of BRG1, the catalytic subunit of the SWI/SNF complex, led to impaired self-renewing ability and dysregulated lineage specification of hESCs. A unique composition of the BRG1-SWI/SNF complex in hESCs was further defined by the presence of BRG1, BAF250A, BAF170, BAF155, BAF53A, and BAF47. Genome-wide expression analyses revealed that BRG1 participated in a broad range of biological processes in hESCs through pathways different from those in mESCs. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that BRG1 played a repressive role in transcriptional regulation by modulating the acetylation levels of H3K27 at the enhancers of lineage-specific genes. Our data thus provide valuable insights into molecular mechanisms by which transcriptional repression affects the self-renewal and differentiation of hESCs.

The transcription factors TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4(+)CD8(+) double-positive (DP) stage are unknown. By specific ablation of these factors in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished the output of CD4(+) T cells and redirected CD4(+) T cells to a CD8(+) T cell fate. The role of TCF-1 and LEF-1 in the CD4-versus-CD8 lineage 'choice' was mediated in part by direct positive regulation of the transcription factor Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused derepression of CD4 expression in T cells committed to the CD8(+) lineage without affecting the expression of Runx transcription factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence Cd4. Thus, TCF-1 and LEF-1 adopted distinct genetic 'wiring' to promote the CD4(+) T cell fate and establish CD8(+) T cell identity.

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.

The metastasis of gastric cancer, one of the most common tumors, has a molecular mechanism that is still largely unclear. Here we investigated the role of possible tumor-suppressor miR-1296-5p in the cell migration and invasion of ERBB2-positive gastric cancer. It found that miR-1296-5p was significantly down-regulated in gastric cancer tissues. Moreover, it was down-regulated in lymph node metastatic gastric cancer tissues compared with non-metastatic gastric cancer tissues. The luciferase activity of ERBB2 3'-untranslated region-based reporters constructed in SNU-216 and NUGC-4 gastric cancer cells suggested that ERBB2 was the target gene of miR-1296-5p. Overexpressed miR-1296-5p reduced its target protein level and Rac1 activation, and inhibited the migration and invasion of SNU-216 and NUGC-4 gastric cancer cells. MiR-1296-5p was down-regulated in ERBB2-positive gastric cancer tissues compared with ERBB2-negative gastric cancer tissues. In ERBB2-positive gastric cancers, the miR-1296-5p expression was suppressed in a majority of metastatic lymph node tissues compared to non-metastatic gastric cancer samples. The migration and invasion of gastric cancer cells was inhibited by miR-1296-5p overexpression or herceptin treatment, and rescued by the overexpression of constitutively active Rac1-Q61L or ERBB2. Taken together, our findings first suggest that miR-1296-5p might be involved in the regulation on the migration and invasion of human gastric cancer cells at least in part via targeting ERBB2/Rac1 signaling pathway.

Sporopollenin is a major component of the pollen exine pattern. In Arabidopsis, acyl-CoA synthetase5 (ACOS5) is involved in sporopollenin precursor biosynthesis. In this study, we identified its orthologue, OsACOS12, in rice (Oryza sativa) and compared the functional conservation of ACOS in rice to Arabidopsis.

Over 100 genetic loci harbor schizophrenia-associated variants, yet how these variants confer liability is uncertain. The CommonMind Consortium sequenced RNA from dorsolateral prefrontal cortex of people with schizophrenia (N = 258) and control subjects (N = 279), creating a resource of gene expression and its genetic regulation. Using this resource, ∼20% of schizophrenia loci have variants that could contribute to altered gene expression and liability. In five loci, only a single gene was involved: FURIN, TSNARE1, CNTN4, CLCN3 or SNAP91. Altering expression of FURIN, TSNARE1 or CNTN4 changed neurodevelopment in zebrafish; knockdown of FURIN in human neural progenitor cells yielded abnormal migration. Of 693 genes showing significant case-versus-control differential expression, their fold changes were ≤ 1.33, and an independent cohort yielded similar results. Gene co-expression implicates a network relevant for schizophrenia. Our findings show that schizophrenia is polygenic and highlight the utility of this resource for mechanistic interpretations of genetic liability for brain diseases.

The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. Here we show that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine.

Genome-wide association studies (GWAS) have identified many loci associated with breast cancer risk. These studies have primarily been conducted in populations of European descent.