Clara Cell 10-kDa Protein Gene Transfection Inhibits NF-κB Activity in Airway Epithelial Cells

Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases.To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10's effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10's effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration.These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation

Non-hematopoietic cells contribute to protective tolerance to Aspergillus fumigatus via a TRIF pathway converging on IDO

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Whereas the pivotal role of dendritic cells in determining the balance between immunopathology and protective immunity to the fungus is well established, we determined that epithelial cells (ECs) also contributes to this balance. Mechanistically, EC-mediated protection occurred through a Toll-like receptor 3/Toll/IL-1 receptor domain-containing adaptor-inducing interferon (TLR3/TRIF)-dependent pathway converging on indoleamine 2,3-dioxygenase (IDO) via non-canonical nuclear factor-?B activation. Consistent with the high susceptibility of TRIF-deficient mice to pulmonary aspergillosis, bone marrow chimeric mice with TRIF unresponsive ECs exhibited higher fungal burdens and inflammatory pathology than control mice, underexpressed the IDO-dependent T helper 1/regulatory T cell (Th1/Treg) pathway and overexpressed the Th17 pathway with massive neutrophilic inflammation in the lungs. Further studies with interferon (IFN)-?, IDO or IL-17R unresponsive cells confirmed the dependency of immune tolerance to the fungus on the IFN-?/IDO/Treg pathway and of immune resistance on the MyD88 pathway controlling the fungal growth. Thus, distinct immune pathways contribute to resistance and tolerance to the fungus, to which the hematopoietic/non-hematopoietic compartments contribute through distinct, yet complementary, roles.We thank Cristina Massi Benedetti for digital art and editing. This work was supported by the Specific Targeted Research Project 'Sybaris' (LSHE-CT-2006), contract number 037899 (FP7) and by the Italian Projects PRIN 2007KLCKP8_004 (to LR) and 2007XYB9T9_001 (to SB). CC and AC were financially supported by fellowships from Fundacao para a Ciencia e Tecnologia, Portugal (contracts SFRH/BD/65962/2009 and SFRH/BPD/46292/2008, respectively)

SProtP: A Web Server to Recognize Those Short-Lived Proteins Based on Sequence-Derived Features in Human Cells

Protein turnover metabolism plays important roles in cell cycle progression, signal transduction, and differentiation. Those proteins with short half-lives are involved in various regulatory processes. To better understand the regulation of cell process, it is important to study the key sequence-derived factors affecting short-lived protein degradation. Until now, most of protein half-lives are still unknown due to the difficulties of traditional experimental methods in measuring protein half-lives in human cells. To investigate the molecular determinants that affect short-lived proteins, a computational method was proposed in this work to recognize short-lived proteins based on sequence-derived features in human cells. In this study, we have systematically analyzed many features that perhaps correlated with short-lived protein degradation. It is found that a large fraction of proteins with signal peptides and transmembrane regions in human cells are of short half-lives. We have constructed an SVM-based classifier to recognize short-lived proteins, due to the fact that short-lived proteins play pivotal roles in the control of various cellular processes. By employing the SVM model on human dataset, we achieved 80.8% average sensitivity and 79.8% average specificity, respectively, on ten testing dataset (TE1-TE10). We also obtained 89.9%, 99% and 83.9% of average accuracy on an independent validation datasets iTE1, iTE2 and iTE3 respectively. The approach proposed in this paper provides a valuable alternative for recognizing the short-lived proteins in human cells, and is more accurate than the traditional N-end rule. Furthermore, the web server SProtP (http://reprod.njmu.edu.cn/sprotp) has been developed and is freely available for users

On systems and control approaches to therapeutic gain

BACKGROUND: Mathematical models of cancer relevant processes are being developed at an increasing rate. Conceptual frameworks are needed to support new treatment designs based on such models. METHODS: A modern control perspective is used to formulate two therapeutic gain strategies. RESULTS: Two conceptually distinct therapeutic gain strategies are provided. The first is direct in that its goal is to kill cancer cells more so than normal cells, the second is indirect in that its goal is to achieve implicit therapeutic gains by transferring states of cancer cells of non-curable cases to a target state defined by the cancer cells of curable cases. The direct strategy requires models that connect anti-cancer agents to an endpoint that is modulated by the cause of the cancer and that correlates with cell death. It is an abstraction of a strategy for treating mismatch repair (MMR) deficient cancers with iodinated uridine (IUdR); IU-DNA correlates with radiation induced cell killing and MMR modulates the relationship between IUdR and IU-DNA because loss of MMR decreases the removal of IU from the DNA. The second strategy is indirect. It assumes that non-curable patient outcomes will improve if the states of their malignant cells are first transferred toward a state that is similar to that of a curable patient. This strategy is difficult to employ because it requires a model that relates drugs to determinants of differences in patient survival times. It is an abstraction of a strategy for treating BCR-ABL pro-B cell childhood leukemia patients using curable cases as the guides. CONCLUSION: Cancer therapeutic gain problem formulations define the purpose, and thus the scope, of cancer process modeling. Their abstractions facilitate considerations of alternative treatment strategies and support syntheses of learning experiences across different cancers

Mapping the Spatio-Temporal Pattern of the Mammalian Target of Rapamycin (mTOR) Activation in Temporal Lobe Epilepsy

Growing evidence from rodent models of temporal lobe epilepsy (TLE) indicates that dysregulation of the mammalian target of rapamycin (mTOR) pathway is involved in seizures and epileptogenesis. However, the role of the mTOR pathway in the epileptogenic process remains poorly understood. Here, we used an animal model of TLE and sclerotic hippocampus from patients with refractory TLE to determine whether cell-type specific activation of mTOR signaling occurs during each stage of epileptogenesis. In the TLE mouse model, we found that hyperactivation of the mTOR pathway is present in distinct hippocampal subfields at three different stages after kainate-induced seizures, and occurs in neurons of the granular and pyramidal cell layers, in reactive astrocytes, and in dispersed granule cells, respectively. In agreement with the findings in TLE mice, upregulated mTOR was observed in the sclerotic hippocampus of TLE patients. All sclerotic hippocampus (n = 13) exhibited widespread reactive astrocytes with overactivated mTOR, some of which invaded the dispersed granular layer. Moreover, two sclerotic hippocampus exhibited mTOR activation in some of the granule cells, which was accompanied by cell body hypertrophy. Taken together, our results indicate that mTOR activation is most prominent in reactive astrocytes in both an animal model of TLE and the sclerotic hippocampus from patients with drug resistant TLE

Fish-Specific Duplicated dmrt2b Contributes to a Divergent Function through Hedgehog Pathway and Maintains Left-Right Asymmetry Establishment Function

Gene duplication is thought to provide raw material for functional divergence and innovation. Fish-specific dmrt2b has been identified as a duplicated gene of the dmrt2a/terra in fish genomes, but its function has remained unclear. Here we reveal that Dmrt2b knockdown zebrafish embryos display a downward tail curvature and have U-shaped somites. Then, we demonstrate that Dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway, because Dmrt2b knockdown reduces target gene expression of Hedgehog signaling, and also impairs slow muscle development and neural tube patterning through Hedgehog signaling. Moreover, the Dmrt2b morphants display defects in heart and visceral organ asymmetry, and, some lateral-plate mesoderm (LPM) markers expressed in left side are randomized. Together, these data indicate that fish-specific duplicated dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway and maintains the common function for left-right asymmetry establishment

MicroRNA-sequence profiling reveals novel osmoregulatory microRNA expression patterns in catadromous eel anguilla marmorata

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression by post-transcriptional repression of mRNAs. Recently, several miRNAs have been confirmed to execute directly or indirectly osmoregulatory functions in fish via translational control. In order to clarify whether miRNAs play relevant roles in the osmoregulation of Anguilla marmorata, three sRNA libraries of A. marmorata during adjusting to three various salinities were sequenced by Illumina sRNA deep sequencing methods. Totally 11,339,168, 11,958,406 and 12,568,964 clear reads were obtained from 3 different libraries, respectively. Meanwhile, 34 conserved miRNAs and 613 novel miRNAs were identified using the sequence data. MiR-10b-5p, miR-181a, miR-26a-5p, miR-30d and miR-99a-5p were dominantly expressed in eels at three salinities. Totally 29 mature miRNAs were significantly up-regulated, while 72 mature miRNAs were significantly down-regulated in brackish water (10‰ salinity) compared with fresh water (0‰ salinity); 24 mature miRNAs were significantly up-regulated, while 54 mature miRNAs were significantly down-regulated in sea water (25‰ salinity) compared with fresh water. Similarly, 24 mature miRNAs were significantly up-regulated, while 45 mature miRNAs were significantly down-regulated in sea water compared with brackish water. The expression patterns of 12 dominantly expressed miRNAs were analyzed at different time points when the eels transferred from fresh water to brackish water or to sea water. These miRNAs showed differential expression patterns in eels at distinct salinities. Interestingly, miR-122, miR-140-3p and miR-10b-5p demonstrated osmoregulatory effects in certain salinities. In addition, the identification and characterization of differentially expressed miRNAs at different salinities can clarify the osmoregulatory roles of miRNAs, which will shed lights for future studies on osmoregulation in fish