Previous studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI). The molecular mechanism of PBEF involvement in the pathogenesis of ALI is still incompletely understood. This study examined the role of PBEF in regulating pulmonary alveolar epithelial cell IL-8 expression and permeability.

The tuberous sclerosis complex (TSC)-mammalian target of rapamycin (mTOR) pathway is a key regulator of cellular metabolism. We used conditional deletion of Tsc1 to address how quiescence is associated with the function of hematopoietic stem cells (HSCs). We demonstrate that Tsc1 deletion in the HSCs drives them from quiescence into rapid cycling, with increased mitochondrial biogenesis and elevated levels of reactive oxygen species (ROS). Importantly, this deletion dramatically reduced both hematopoiesis and self-renewal of HSCs, as revealed by serial and competitive bone marrow transplantation. In vivo treatment with an ROS antagonist restored HSC numbers and functions. These data demonstrated that the TSC-mTOR pathway maintains the quiescence and function of HSCs by repressing ROS production. The detrimental effect of up-regulated ROS in metabolically active HSCs may explain the well-documented association between quiescence and the "stemness" of HSCs.

The mammalian target of rapamycin (mTOR) promotes anabolic cellular processes in response to growth factors and metabolic cues. The TSC1 and TSC2 tumor suppressors are major upstream inhibitory regulators of mTOR signaling. Mice with Rip2/Cre-mediated deletion of Tsc1 (Rip-Tsc1cKO mice) developed hyperphagia and obesity, suggesting that hypothalamic disruption (for which Rip2/Cre is well known) of Tsc1 may dysregulate feeding circuits via mTOR activation. Indeed, Rip-Tsc1cKO mice displayed increased mTOR signaling and enlarged neuron cell size in a number of hypothalamic populations, including Pomc neurons. Furthermore, Tsc1 deletion with Pomc/Cre (Pomc-Tsc1cKO mice) resulted in dysregulation of Pomc neurons and hyperphagic obesity. Treatment with the mTOR inhibitor, rapamycin, ameliorated the hyperphagia, obesity, and the altered Pomc neuronal morphology in developing or adult Pomc-Tsc1cKO mice, and cessation of treatment reinstated these phenotypes. Thus, ongoing mTOR activation in Pomc neurons blocks the catabolic function of these neurons to promote nutrient intake and increased adiposity.

The tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor. However, the inhibitory activities of these variants have not yet been compared in the same cells. Protein therapy is known to have several advantages over gene therapy. Thus, investigation of the exogenous protein molecule of the most effective suppressor may yield meaningful information regarding protein-based cancer therapy.

Hemopoietic system derived progenitor cells with mesenchymal features have been identified including CD14(+) monocyte-derived progenitors. However, it is unclear whether there are mesenchyme derived progenitors with hematopoietic potential. Herein, we identified a novel CD14(-) cell-derived population with both mesenchymal and hematopoietic features in rat peripheral blood, and this cell population is different from the CD14(+) monocyte-derived progenitors but designated peripheral blood multipotential mesenchymal progenitors (PBMMPs). Phenotype analysis demonstrated expression of mesenchymal markers in PBMMPs including BMPRs, Endoglin/CD105, Fibronectin (Fn), Vimentin (Vim), Collagen (Col) I/II/III along with hematopoietic marker CD34. CD14(+) cell-derived population shared the same characteristics with CFs. In mixed culture of CD14(+) and CD14(-) cells, PBMMPs were a predominant component and expressed CD29(high), CD73(high), CD34(high), CD45(low) and CD90. Except for the value of mixed T lymphocytes and CD14(+) cell-derived population, hematopoietic characters of cultured PBMMPs were indicated by CD14(-)/CD34(+)/CD45(-)/CD90(+). The mesenchymal origin was further confirmed by comparing PBMMPs with bone marrow stromal cells. Finally, we transplanted PBMMPs into a skin wound model, and results showed the specific potential of PBMMPs in not only extracellular matrix secretion but epidermal regeneration. This study provides evidence that peripheral blood contains common hematopoietic-mesenchymal progenitors from both hematopoietic and mesenchymal lineages, and CD34(+) mesenchymal progenitors are a possible alternative source of epidermal cells in wound healing.

Protein kinase C (PKC) is involved in a wide array of cellular processes such as cell proliferation, differentiation and apoptosis. Phosphorylation of both turn motif (TM) and hydrophobic motif (HM) are important for PKC function. Here, we show that the mammalian target of rapamycin complex 2 (mTORC2) has an important function in phosphorylation of both TM and HM in all conventional PKCs, novel PKCepsilon as well as Akt. Ablation of mTORC2 components (Rictor, Sin1 or mTOR) abolished phosphorylation on the TM of both PKCalpha and Akt and HM of Akt and decreased HM phosphorylation of PKCalpha. Interestingly, the mTORC2-dependent TM phosphorylation is essential for PKCalpha maturation, stability and signalling. Our study demonstrates that mTORC2 is involved in post-translational processing of PKC by facilitating TM and HM phosphorylation and reveals a novel function of mTORC2 in cellular regulation.

A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

Streptococcus suis serotype 2 (SS2) is an emerging human pathogen worldwide. A large outbreak occurred in the summer of 2005 in China. Serum samples from this outbreak revealed that levels of the main proinflammatory cytokines were significantly higher in patients with streptococcal toxic-shock-like syndrome (STSLS) than in patients with meningitis only. However, the mechanism underlying the cytokine storm in STSLS caused by SS2 remained unclear. In this study, we found that suilysin (SLY) is the main protein inflammatory stimulus of SS2 and that native SLY (nSLY) stimulated cytokines independently of its haemolytic ability. Interestingly, a small amount of SLY (Å Mol/L) induced strong, long-term TNF-α release from human PBMCs. We also found that nSLY stimulated TNF-α in wild-type macrophages but not in macrophages from mice that carried a spontaneous mutation in TLR4 (P712H). We demonstrated for the first time that SLY stimulates immune cells through TLR4. In addition, the Myd88 adaptor-p38-MAPK pathway was involved in this process. The present study suggested that the TLR4-dependent inflammatory responses induced by SLY in host might contribute to the STSLS caused by SS2 and that p38-MAPK could be used as a target to control the release of excess TNF-α induced by SS2.

Amyloid plaques composed of β-amyloid (Aβ) protein are a pathological hallmark of Alzheimer's disease. We here report the generation and characterization of a novel transgenic mouse model of Aβ toxicity. The rTg9191 mice harbor a transgene encoding the 695 amino-acid isoform of human amyloid precursor protein (APP) with the Swedish and London mutations (APPNLI) linked to familial Alzheimer's disease, under the control of a tetracycline-response element, as well as a transgene encoding the tetracycline transactivator, under the control of the promoter for calcium-calmodulin kinase IIα. In these mice, APPNLI is expressed at a level four-fold that of endogenous mouse APP and its expression is restricted to forebrain regions. Transgene expression was suppressed by 87% after two months of doxycycline administration. Histologically, we showed that (1) Aβ plaques emerged in cerebral cortex and hippocampus as early as 8 and 10.5-12.5 months of age, respectively; (2) plaque deposition progressed in an age-dependent manner, occupying up to 19% of cortex at ~25 months of age; and (3) neuropathology--such as abnormal neuronal architecture, tau hyperphosphorylation and misfolding, and neuroinflammation--was observed in the vicinity of neuritic plaques. Biochemically, we determined total Aβ production at varied ages of mice, and we showed that mice produced primarily fibrillar Aβ assemblies recognized by conformation-selective OC antibodies, but few non-fibrillar oligomers (e.g., Aβ*56) detectable by A11 antibodies. Finally, we showed that expression of the tetracycline transactivator resulted in reduced brain weight and smaller dentate-gyrus size. Collectively, these data indicate that rTg9191 mice may serve as a model for studying the neurological effects of the fibrillar Aβ assemblies in situ.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

The cellular mechanisms controlling infection-induced emergency granulopoiesis are poorly defined. Here we found that reactive oxygen species (ROS) concentrations in the bone marrow (BM) were elevated during acute infection in a phagocytic NADPH oxidase-dependent manner in myeloid cells. Gr1(+) myeloid cells were uniformly distributed in the BM, and all c-kit(+) progenitor cells were adjacent to Gr1(+) myeloid cells. Inflammation-induced ROS production in the BM played a critical role in myeloid progenitor expansion during emergency granulopoiesis. ROS elicited oxidation and deactivation of phosphatase and tensin homolog (PTEN), resulting in upregulation of PtdIns(3,4,5)P3 signaling in BM myeloid progenitors. We further revealed that BM myeloid cell-produced ROS stimulated proliferation of myeloid progenitors via a paracrine mechanism. Taken together, our results establish that phagocytic NADPH oxidase-mediated ROS production by BM myeloid cells plays a critical role in mediating emergency granulopoiesis during acute infection.

A system of novel nanoparticles of star-shaped cholic acid-core polylactide-d-α-tocopheryl polyethylene glycol 1000 succinate (CA-PLA-TPGS) block copolymer was developed for paclitaxel delivery for breast cancer treatment, which demonstrated superior in vitro and in vivo performance in comparison with paclitaxel-loaded poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles and linear PLA-TPGS nanoparticles. The paclitaxel- or couramin 6-loaded nanoparticles were fabricated by a modified nanoprecipitation method and then characterized in terms of size, surface charge, surface morphology, drug encapsulation efficiency, and in vitro drug release. The CA-PLA-TPGS nanoparticles were found to be spherical in shape with an average size of around 120 nm. The nanoparticles were found to be stable, showing no change in the particle size and surface charge during 90-day storage of the aqueous solution. The release profiles of the paclitaxel-loaded nanoparticles exhibited typically biphasic release patterns. The results also showed that the CA-PLA-TPGS nanoparticles have higher antitumor efficacy than the PLA-TPGS nanoparticles and PLGA nanoparticles in vitro and in vivo. In conclusion, such nanoparticles of star-shaped cholic acid-core PLA-TPGS block copolymer could be considered as a potentially promising and effective strategy for breast cancer treatment.

Autophagy is the primary cellular catabolic program activated in response to nutrient starvation. Initiation of autophagy, particularly by amino-acid withdrawal, requires the ULK kinases. Despite its pivotal role in autophagy initiation, little is known about the mechanisms by which ULK promotes autophagy. Here we describe a molecular mechanism linking ULK to the pro-autophagic lipid kinase VPS34. Following amino-acid starvation or mTOR inhibition, the activated ULK1 phosphorylates Beclin-1 on Ser 14, thereby enhancing the activity of the ATG14L-containing VPS34 complexes. The Beclin-1 Ser 14 phosphorylation by ULK is required for full autophagic induction in mammals and this requirement is conserved in Caenorhabditis elegans. Our study reveals a molecular link from ULK1 to activation of the autophagy-specific VPS34 complex and autophagy induction.

Degradation of p12 subunit of human DNA polymerase delta (Pol δ) that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi), restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.

RNA-Sequencing (RNA-seq) experiments have been popularly applied to transcriptome studies in recent years. Such experiments are still relatively costly. As a result, RNA-seq experiments often employ a small number of replicates. Power analysis and sample size calculation are challenging in the context of differential expression analysis with RNA-seq data. One challenge is that there are no closed-form formulae to calculate power for the popularly applied tests for differential expression analysis. In addition, false discovery rate (FDR), instead of family-wise type I error rate, is controlled for the multiple testing error in RNA-seq data analysis. So far, there are very few proposals on sample size calculation for RNA-seq experiments.

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

SAMHD1 hydrolyzes 2'-deoxynucleoside-5'-triphosphates (dNTPs) into 2'-deoxynucleosides and inorganic triphosphate products. In this paper, we evaluated the impact of 2' sugar moiety substitution for different nucleotides on being substrates for SAMHD1 and mechanisms of actions for the results. We found that dNTPs ((2'R)-2'-H) are only permissive in the catalytic site of SAMHD1 due to L150 exclusion of (2'R)-2'-F and (2'R)-2'-OH nucleotides. However, arabinose ((2'S)-2'-OH) nucleoside-5'-triphosphates analogs are permissive to bind in the catalytic site and be hydrolyzed by SAMHD1. Moreover, when the (2'S)-2' sugar moiety is increased to a (2'S)-2'-methyl as with the SMDU-TP analog, we detect inhibition of SAMHD1's dNTPase activity. Our computational modeling suggests that (2'S)-2'-methyl sugar moiety clashing with the Y374 of SAMHD1. We speculate that SMDU-TP mechanism of action requires that the analog first docks in the catalytic pocket of SAMHD1 but prevents the A351-V378 helix conformational change from being completed, which is needed before hydrolysis can occur. Collectively we have identified stereoselective 2' substitutions that reveal nucleotide substrate specificity for SAMHD1, and a novel inhibitory mechanism for the dNTPase activity of SAMHD1. Importantly, our data is beneficial for understanding if FDA-approved antiviral and anticancer nucleosides are hydrolyzed by SAMHD1 in vivo.

Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

To characterize the microRNAs that contribute to the development of brace root, Solexa high-throughput sequencing of three libraries derived from tissues of node (N), nodes with just-emerged brace roots (NR), and nodes with just-emerged brace roots after IAA treatment (NRI) was performed. Total 650,793, 957,303 and 1,082,948 genome-matched unique reads were obtained in N, NR and NRI libraries, respectively. Further analysis confirmed the authenticity of 137 known miRNAs and the discovery of 159 novel miRNAs in maize. 14 conserved and 16 novel miRNAs differentially expressed in brace root, as well as 15 target genes, were identified and validated by qRT-PCR during maize brace root development. Moreover, we identified 9 miRNA precursor-matched novel sRNAs that may form miRNA clusters, as well as 24 nt siRNAs in the three libraries. In addition, we suggest that auxin represent a regulator in brace root development and can be regulated at the posttranscriptional level by miRNAs.

The chimeric fusion protein, AML1-ETO, generated by translocation of t(8;21), abnormally recruits histone deacetylase (HDAC) to the promoters of AML1 target genes, resulting in transcriptional repression of the target genes and development of t(8;21) acute myeloid leukemia. Abnormal expression of cyclin-dependent kinase inhibitors, especially p21, is considered a possible mechanism of the arrested maturation and differentiation seen in leukemia cells. A new generation of HDAC inhibitors is becoming an increasing focus of attention for their ability to induce differentiation and apoptosis in tumor cells and to block the cell cycle. Our previous research had demonstrated that valproic acid induces G0/G1 arrest of Kasumi-1 cells in t(8;21) acute myeloid leukemia. In this study, we further confirmed that valproic acid inhibits the growth of Kasumi-1 cells in a murine xenograft tumor model, and that this occurs via upregulation of histone acetylation in the p21 promoter region, enhancement of p21 expression, suppression of phosphorylation of retinoblastoma protein, blocking of transcription activated by E2F, and induction of G0/G1 arrest.