BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes

Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperone-assisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis

Clinical significance of PTEN and p-Akt co-expression in HER2-positive metastatic breast cancer patients treated with trastuzumab-based therapies

Objective: The phosphatase and tensine homologue gene (PTEN) plays a crucial role in proliferation and survival of cancer cells by antagonizing the function of phosphatidylinositol 3'-kinase (PI3K), which, in turn, results in decreased Akt activity. We investigated the clinical impact of the expression of PTEN, p-Akt and PI3K in HER2-positive metastatic breast cancer (MBC) patients treated with trastuzumab-based therapies. Methods: Seventy-three patients treated with trastuzumab-based therapies were included and followed prospectively. PTEN, p-Akt and PI3K expression was determined by immunohistochemistry. Results: PTEN, p-Akt and PI3K resulted positive in 48%, 71% and 46.5% of patients, respectively. A significant correlation between PTEN and p-Akt (kappa 0.22, p = 0.03) and p-Akt and PI3K (kappa 0.20, p = 0.05) was observed. PTEN-positive patients had a progression-free survival (PFS) longer than PTEN-negative ones (p = 0.06). When grouped together, patients co-expressing PTEN and p-Akt had a statistically significant longer PFS as compared to the rest of patients (p = 0.01). At the multivariate analysis, PTEN and p-Akt co-expression was an independent predictor of lower risk of progression (hazard ratio 0.53, p = 0.05). Conclusion: In HER2-positive MBC, basal co-expression of PTEN and p-Akt might identify those patients who are more likely to benefit from trastuzumab-based therapies

Sphingosine-1 phosphate induces cAMP/PKA-independent phosphorylation of the cAMP response element-binding protein (CREB) in granulosa cells

Background and aims: Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 \u3bcM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. Conclusions: This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P\u2013S1PR axis may cooperate with gonadotropins in modulating follicle development

Prognostic impact of alternative splicing-derived hMENA isoforms in resected, node-negative, non-small-cell lung cancer

Risk assessment and treatment choice remain a challenge in early non-small-cell lung cancer (NSCLC). Alternative splicing is an emerging source for diagnostic, prognostic and therapeutic tools. Here, we investigated the prognostic value of the actin cytoskeleton regulator hMENA and its isoforms, hMENA(11a) and hMENA Delta v6, in early NSCLC. The epithelial hMENA(11a) isoform was expressed in NSCLC lines expressing E-CADHERIN and was alternatively expressed with hMENA Delta v6. Enforced expression of hMENA Delta v6 or hMENA(11a) increased or decreased the invasive ability of A549 cells, respectively. hMENA isoform expression was evaluated in 248 node-negative NSCLC. High pan-hMENA and low hMENA(11a) were the only independent predictors of shorter disease-free and cancer-specific survival, and low hMENA(11a) was an independent predictor of shorter overall survival, at multivariate analysis. Patients with low pan-hMENA/high hMENA(11a) expression fared significantly better (P <= 0.0015) than any other subgroup. Such hybrid variable was incorporated with T-size and number of resected lymph nodes into a 3-class-risk stratification model, which strikingly discriminated between different risks of relapse, cancer-related death, and death. The model was externally validated in an independent dataset of 133 patients. Relative expression of hMENA splice isoforms is a powerful prognostic factor in early NSCLC, complementing clinical parameters to accurately predict individual patient risk

An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle Cell Disease Phenotype

Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling HBB variant is a potential treatment for sickle cell disease (SCD). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling HBB transgene (HBBAS3), a minimal HBB promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCD patients were transduced with LVs containing either HS2 and HS3 (\u3b2-AS3) or HS2, HS3, and HS4 (\u3b2-AS3 HS4). The inclusion of the HS4 element drastically reduced vector titer and infectivity in HSPCs, with negligible improvement of transgene expression. Conversely, the LV containing only HS2 and HS3 was able to efficiently transduce SCD bone marrow and Plerixafor-mobilized HSPCs, with anti-sickling HBB representing up to 3c60% of the total HBB-like chains. The expression of the anti-sickling HBB and the reduced incorporation of the \u3b2S-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling HBB transgene ameliorating the SCD cell phenotype

Castel di Sangro-Scontrone field camp – structural and applied geomorphology

The Geomorphological Field Camp 2014 in the Castel di Sangro-Scontrone area is the result of geological and geomorphological teaching field work activities carried out in Central Italy by a group of 23 students attending the Structural Geomorphology and Applied Geomorphology courses (Master's Degree in Geological Science and Technology of the Università degli Studi ‘G. d'Annunzio’ Chieti-Pescara, Italy, Department of Engineering and Geology). The Field Camp 2014 was organized in May 2014, following regular classes held during the fall term. General activities for the field camp were developed over four main stages: (1) preliminary analysis of the regional geological and geomorphological setting of the area; (2) preliminary activities for the analysis of the local area (orography, hydrography and photogeology investigations, and geographical information system processing); (3) field work, focused on the analysis of a specific issue concerning structural geomorphology or applied geomorphology (e.g. landscape evolution, river channel change, landslide distribution, and flood hazard); and (4) post-field work production of the map. Finally, the fundamental role of field work in the analysis of landscape and in land management was outlined: indeed, the overall field camp enhanced the crucial role of field-based learning for young geomorphologists in order to acquire a strong sensitivity to geomorphological processes and landscape evolution

Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR

miR-10b*, a master inhibitor of the cell cycle, is down-regulated in human breast tumours

Deregulated proliferation is a hallmark of cancer cells. Here, we show that microRNA-10b* is a master regulator of breast cancer cell proliferation and is downregulated in tumoural samples versus matched peritumoural counterparts. Two canonical CpG islands (5kb) upstream from the precursor sequence are hypermethylated in the analysed breast cancer tissues. Ectopic delivery of synthetic microRNA-10b* in breast cancer cell lines or into xenograft mouse breast tumours inhibits cell proliferation and impairs tumour growth in vivo, respectively. We identified and validated in vitro and in vivo three novel target mRNAs of miR-10b* (BUB1, PLK1 and CCNA2), which play a remarkable role in cell cycle regulation and whose high expression in breast cancer patients is associated with reduced disease-free survival, relapse-free survival and metastasis-free survival when compared to patients with low expression. This also suggests that restoration of microRNA-10b* expression might have therapeutic promise

Lack of functional pnp gene results in increased cell aggregation and poly-N-acetylglucosamine (PNAG) production in Escherichia coli C

The exoribonuclease polynucleotide phosphorylase (PNPase) encoded by the pnp gene, is a major player in RNA processing. In Escherichia coli C the lack of a functional pnp gene results in macroscopic aggregation in glucose-based medium. In order to identify specific adhesion factors involved in cell aggregation, we created a set of isogenic derivatives deficient in the production of curli, cellulose, colanic acid and poly-N-acetylglucosamine (PNAG). Only inactivation of the PNAG biosynthetic genes pgaA and pgaC suppressed cell aggregation phenotype in the pnp mutant, suggesting that PNAG mediated cell aggregation in the absence of PNPase. Real Time PCR experiments showed that in the pnp- strain pgaA gene expression is 12-fold higher than in the wild type. Previous observations suggest that PNAG biosynthetic operon pgaABCD is regulated at post transcriptional level through a mechanism involving the operon 5\u2019-UTR. Thus, in order to test whether PNPase directly regulates pgaABCD transcripts, we constructed plasmids in which the luxAB reporter was placed under the control either of the pgaABCD regulatory elements (promoter and 5\u2019-UTR) or of the promoter region alone (\uf044UTR construct). Our results show that the presence of the 5\u2019-UTR is necessary for PNPase-dependent regulation. We also compared by SDS-PAGE the outer-membrane associated proteins (OMPs) expressed by pnp+ and pnp- E. coli C. Consistent with our previous observations, the PgaA protein, necessary for PNAG secretion, was only found in the outer membrane fraction of the pnp- strain. Finally, PNAG overproduction in the pnp- strain was verified by dot-blot analysis with anti-PNAG specific antibodies. Our results clearly show that lack of PNPase positively affects PNAG production, suggesting that PNPase is a negative regulator of pgaABCD operon expression. The molecular mechanism of pgaABCD regulation by PNPase is currently under investigation