Liver osteopontin is required to prevent the progression of age‐related nonalcoholic fatty liver disease

Osteopontin (OPN), a senescence‐associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age‐dependent hepatosteatosis. Thus, we investigated the role of OPN in the age‐related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild‐type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN‐deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN‐KO mice liver were associated with the decrease of 78 kDa glucose‐regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53‐OPN axis is required to inhibit the onset of age‐related hepatosteatosis.     

Structural insights into cognate versus near-cognate discrimination during decoding

The structural basis of the tRNA selection process is investigated by cryo-electron microscopy of ribosomes programmed with UGA codons and incubated with ternary complex (TC) containing the near-cognate Trp-tRNA(Trp) in the presence of kirromycin. Going through more than 350 000 images and employing image classification procedures, we find ～8% in which the TC is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near-cognate aa-tRNA with respect to elongation factor Tu (EF-Tu) and the ribosome, as well as the domain movements of the ribosome. One of the interesting findings is that near-cognate tRNA's acceptor stem region is flexible and CCA end becomes disordered. The data bring direct structural insights into the induced-fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa-tRNA and EF-Tu and comparison with the cognate case (UGG codon) offers clues on how the conformational signals conveyed to the GTPase differ in the two cases.     

ERCC1 (excision repair cross-complementing 1) expression in pT2 gallbladder cancer is a prognostic factor

Gallbladder cancer (GBC) is the main cause of death by malignant tumour in women in Chile. There is no information regarding the role of excision repair cross-complementing group 1 (ERCC1) in GBC. Our aim is to determine the expression and significance of ERCC1 as a prognostic factor in GBC. Tissue microarrays were prepared using 200 surgically resected GBCs and 50 non-malignant gallbladders as controls. In 190 cases, ERCC1 was determined by immunohistochemistry. The correlation between ERCC1 expression and GBC pathological characteristics and patient survival were analysed. Ninety-five percent of the non-malignant gallbladder epithelia showed intense and diffuse ERCC1 expression. GBC cases showed ERCC1 expression in the tumour cells in 100/190 (53%) cases. The best differentiated tumours showed significantly greater expression than the less differentiated (p<0.05). Patients with ERCC1-positive status with subserosal carcinomas (pT2) had significantly better survival than ERCC1-negative patients at 20 and 60 months of follow-up (p=0.005), and the probability of dying was 6 times lower for ERCC1-positive than for ERCC1-negative patients. Our preliminary results show that chole-cystectomised patients with GBC in stage pT2 and with ERCC1 expression have significantly better survival than patients at the same stage that did not present ERCC1 expression.     

Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition

Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA-snRNA-interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.     

TET2 mutations are associated with specific 5-methylcytosine and 5-hydroxymethylcytosine profiles in patients with chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has been recently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile.     

Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.     

Quasi-atomic model of bacteriophage t7 procapsid shell: insights into the structure and evolution of a basic fold

The existence of similar folds among major structural subunits of viral capsids has shown unexpected evolutionary relationships suggesting common origins irrespective of the capsids' host life domain. Tailed bacteriophages are emerging as one such family, and we have studied the possible existence of the HK97-like fold in bacteriophage T7. The procapsid structure at approximately 10 A resolution was used to obtain a quasi-atomic model by fitting a homology model of the T7 capsid protein gp10 that was based on the atomic structure of the HK97 capsid protein. A number of fold similarities, such as the fitting of domains A and P into the L-shaped procapsid subunit, are evident between both viral systems. A different feature is related to the presence of the amino-terminal domain of gp10 found at the inner surface of the capsid that might play an important role in the interaction of capsid and scaffolding proteins.