IWS1 is an RNA-polymerase II (RNAPII)-associated transcription elongation factor whose biological functions are poorly characterized. To shed some light on the function of this protein at the organismal level, we performed a systematic tissue analysis of its expression and generated Iws1-deficient mice. A thorough immunohistochemical characterization shows that IWS1 protein is present in the nucleus of all cells in most of the examined tissues, with few notable exceptions. We also report that ablation of Iws1 consistently causes lethality at the pre-implantation stage with high expression of the gene in fertilized oocytes. In summary, we are providing evidence that Iws1 is expressed in all adult organs and it is an essential gene for mouse embryonic development.

The essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function.

Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect, resulting from the nonspecific cytoablative actions of chemoagents, including 5-fluorouracil (5-FU) and irinotecan (CPT-11). Preventive strategies are urgently needed for the predictable CIM. Previously, we have demonstrated an important role of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) in the prevention of cyclophosphamide-induced mucositis in mice. In this study, the preventive role of rhIL-1Ra was further evaluated in 5-FU- and CPT-11-induced mucositis mouse models. rhIL-1Ra pretreatment reduced the incidence, severity, and duration of chemotherapy-induced diarrhea, through attenuating crypt apoptosis and improving crypt survival in wild-type mice, but not in IL-1RI(-/-), p53(-/-), and p21(-/-) mice. Further studies demonstrated that rhIL-1Ra promoted the cell cycle arrest of intestinal crypt epithelia (ICE) through elevating the cellular level of p21(WAF1) and p27(KIP1), which was abolished in IL-1RI(-/-) and p53(-/-) mice, and in p21(WAF1) and p27(KIP1) silenced IEC-6 cells. Importantly, the tumor growth and sensitivity to chemotherapy were not affected by rhIL-1Ra in cultures of tumor cell lines and in a syngeneic tumor-transplantation mouse model. The present study demonstrated that rhIL-1Ra effectively and specifically protected ICE from chemotoxicity through reversible reduction of the basal level of IL-1 signaling to promote normal cell cycle arrest, but not tumor cells. Our findings support the clinical development of rhIL-1Ra in the prevention of CIM.

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. The temporally and spatially controlled expression of the EF1-LSL-tTA knockin and activation of tTA-driven responder transgenes was tested using four transgenic lines that express Cre under tissue-specific promoters of the pancreas, mammary gland and other secretory tissues, as well as an interferon-inducible promoter. In all models, the endogenous Eef1a1 promoter facilitated a cell-type-specific activation of target genes at high levels without exogenous enhancer elements. The applicability of the EF1-LSL-tTA strain for biological experiments was tested in two studies related to mammary gland development and tumorigenesis. First, we validated the crucial role of active STAT5 as a survival factor for functionally differentiated epithelial cells by expressing a hyperactive STAT5 mutant in the mammary gland during postlactational remodeling. In a second experiment, we assessed the ability of the EF1-tTA to initiate tumor formation through upregulation of mutant KRAS. The collective results show that the EF1-LSL-tTA knockin line is a versatile genetic tool that can be applied to constitutively express transgenes in specific cell types to examine their biological functions at defined developmental stages.

This study identified an insulin-like peptide (ILP) in Macrobrachium rosenbergii termed Mr-ILP and further investigated its function through glucose injection and RNAi. With the analysis of five other glucose metabolism related genes, this study shed light on the molecular mechanism of carbohydrate metabolism in crustaceans. Mr-ILP shared the typical skeleton with six conserved cysteine and mainly expressed in neuroendocrine system. In M. rosenbergii, the elevated hemolymph glucose concentration after glucose injection returned to basal levels in short time, implying an efficient regulatory system in carbohydrate metabolism. Hyperglycemic related genes answered the elevated hemolymph glucose concentration quickly with significant decreased expression level, while Mr-ILP showed delayed response. Instead, glycolysis increased after glucose injection, which indicated glycolysis might play an important role in lowering the abnormally high glucose level. In vivo silencing of Mr-ILP, by injecting the prawns with double-stranded RNA (dsRNA) for 21 days reduced its expression by approximately 75%. Accordingly, glycogen synthase decreased and the trehalose and glycogen level in the hepatopancreas were significantly reduced, indicating the function of Mr-ILP in oligosaccharide and polysaccharide accumulation. When Mr-ILP was silenced, the expression of hyperglycemic related genes were enhanced, but the hemolymph glucose level was not elevated significantly, which might attribute to the increased glycolysis to keep a balanced glucose level in hemolymph.

Type 2 diabetes (T2D) is associated with a strongly increased risk for restenosis after angioplasty driven by proliferation of vascular smooth muscle cells (VSMCs). Here, we sought to determine whether and how mitochondrial dysfunction in T2D drives VSMC proliferation with a focus on ROS and intracellular [Ca 2+ ] that both drive cell proliferation, occur in T2D and are regulated by mitochondrial activity.

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans.

Two-pore K(+) channels have emerged as potential targets to selectively regulate cardiac cell membrane excitability; however, lack of specific inhibitors and relevant animal models has impeded the effort to understand the role of 2-pore K(+) channels in the heart and their potential as a therapeutic target. The objective of this study was to determine the role of mechanosensitive 2-pore K(+) channel family member TREK-1 in control of cardiac excitability.

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.