BBLN-1 is essential for intermediate filament organization and apical membrane morphology

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C. elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C. elegans intestine, BBLN recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1 in early larvae. In mouse intestinal organoids, BBLN localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals

The centriolar satellite protein Cfap53 facilitates formation of the zygotic microtubule organizing center in the zebrafish embryo

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation

SPOP Deregulation Improves the Radiation Response of Prostate Cancer Models by Impairing DNA Damage Repair

Speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is the most commonly mutated gene in prostate cancer (PCa). Recent evidence reports a role of SPOP in DNA damage response (DDR), indicating a possible impact of SPOP deregulation on PCa radiosensitivity. This study aimed to define the role of SPOP deregulation (by gene mutation or knockdown) as a radiosensitizing factor in PCa preclinical models. To express WT or mutant (Y87N, K129E and F133V) SPOP, DU145 and PC-3 cells were transfected with pMCV6 vectors. Sensitivity profiles were assessed using clonogenic assay and immunofluorescent staining of γH2AX and RAD51 foci. SCID xenografts were treated with 5 Gy single dose irradiation using an image-guided small animal irradiator. siRNA and miRNA mimics were used to silence SPOP or express the SPOP negative regulator miR-145, respectively. SPOP deregulation, by either gene mutation or knockdown, consistently enhanced the radiation response of PCa models by impairing DDR, as indicated by transcriptome analysis and functionally confirmed by decreased RAD51 foci. SPOP silencing also resulted in a significant downregulation of RAD51 and CHK1 expression, consistent with the impairment of homologous recombination. Our results indicate that SPOP deregulation plays a radiosensitizing role in PCa by impairing DDR via downregulation of RAD51 and CHK1. View Full-Tex

Pattern of care and effectiveness of treatment for glioblastoma patients in the real world: Results from a prospective population-based registry. Could survival differ in a high-volume center?

BACKGROUND: As yet, no population-based prospective studies have been conducted to investigate the incidence and clinical outcome of glioblastoma (GBM) or the diffusion and impact of the current standard therapeutic approach in newly diagnosed patients younger than aged 70 years. METHODS: Data on all new cases of primary brain tumors observed from January 1, 2009, to December 31, 2010, in adults residing within the Emilia-Romagna region were recorded in a prospective registry in the Project of Emilia Romagna on Neuro-Oncology (PERNO). Based on the data from this registry, a prospective evaluation was made of the treatment efficacy and outcome in GBM patients. RESULTS: Two hundred sixty-seven GBM patients (median age, 64 y; range, 29-84 y) were enrolled. The median overall survival (OS) was 10.7 months (95% CI, 9.2-12.4). The 139 patients 64aged 70 years who were given standard temozolomide treatment concomitant with and adjuvant to radiotherapy had a median OS of 16.4 months (95% CI, 14.0-18.5). With multivariate analysis, OS correlated significantly with KPS (HR = 0.458; 95% CI, 0.248-0.847; P = .0127), MGMT methylation status (HR = 0.612; 95% CI, 0.388-0.966; P = .0350), and treatment received in a high versus low-volume center (HR = 0.56; 95% CI, 0.328-0.986; P = .0446). CONCLUSIONS: The median OS following standard temozolomide treatment concurrent with and adjuvant to radiotherapy given to (72.8% of) patients aged 6470 years is consistent with findings reported from randomized phase III trials. The volume and expertise of the treatment center should be further investigated as a prognostic factor

Connecting the neuronal proteome: Unraveling protein dynamics in neurons using Mass Spectrometry-based proteomics

Similarly to the amazon rainforest which is formed by a multitude of different trees, many of them growing around and on top of each other with their branches and roots tightly interconnected, the human brain is composed by approximately 100 billion neurons organized into a complex matrix of connections and packed into highly specialized layers. In order to establish and maintain neuronal connections, neurons must be able to sort and deliver proteins to specific compartments with extreme precision. In fact, the protein composition of each single neuronal compartment not only determines its unique architectural morphology and function but has also a more profound effect on the formation of the neuronal network which is the essence of our brain. In this perspective, it is essential to study the primary building blocks of life, the proteins, alongside with their interactions and the molecular mechanisms regulating their functions. A key concept of modern biology is that proteins participate in complex, interconnected networks, rather than linear pathways. In the field of neurobiology, proteomics has recently started to showcase its full potential as a powerful methodology able to outperform classical biochemical approaches by allowing a more global understanding of protein dynamics and a more detailed characterization of intracellular protein networks and complexes. This thesis describes our efforts in advancing the understanding of selected mechanisms in neurons by using Mass Spectrometry (MS)-based proteomics applications. Latest MS techniques have been widely used to investigate neuronal differentiation, kinesin-mediated neuronal transport and protein-interaction networks in physiological or pathological condition

Development and Validation of a New Tool to Improve the Accuracy of the Hospital Mass-Casualty Incident Response Plan Activation: The PEMAAF Score

Introduction: Effective response to a mass-casualty incident (MCI) entails the activation of hospital MCI plans. Unfortunately, there are no tools available in the literature to support hospital responders in predicting the proper level of MCI plan activation. This manuscript describes the scientific-based approach used to develop, test, and validate the PEMAAF score (Proximity, Event, Multitude, Overcrowding, Temporary Ward Reduction Capacity, Time Shift Slot [Prossimita, Evento, Moltitudine, Affollamento, Accorpamento, Fascia Oraria], a tool able to predict the required level of hospital MCI plan activation and to facilitate a coordinated activation of a multi-hospital network.Methods: Three study phases were performed within the Metropolitan City of Milan, Italy: (1) retrospective analysis of past MCI after action reports (AARs); (2) PEMAAF score development; and (3) PEMAAF score validation. The validation phase entailed a multi-step process including two retrospective analyses of past MCIs using the score, a focus group discussion (FGD), and a prospective simulation-based study. Sensitivity and specificity of the score were analyzed using a regression model, Spearman's Rho test, and receiver operating characteristic/ROC analysis curves.Results: Results of the retrospective analysis and FGD were used to refine the PEMAAF score, which included six items-Proximity, Event, Multitude, Emergency Department (ED) Overcrowding, Temporary Ward Reduction Capacity, and Time Shift Slot-allowing for the identification of three priority levels (score of 5-6: green alert; score of 7-9: yellow alert; and score of 10-12: red alert). When prospectively analyzed, the PEMAAF score determined most frequent hospital MCI plan activation (>10) during night and holiday shifts, with a score of 11 being associated with a higher sensitivity system and a score of 12 with higher specificity.Conclusions: The PEMAAF score allowed for a balanced and adequately distributed response in case of MCI, prompting hospital MCI plan activation according to real needs, taking into consideration the whole hospital response network

Hospital Surge Capacity during Expo 2015 in Milano, Italy

IntroductionHospital Acute Care Surge Capacity (HACSC), Hospital Acute Care Surge Threshold (HACST), and Total Hospital Capacity (THC) are scales that were developed to quantify surge capacity in the event of a multiple-casualty incident (MCI). These scales take into consideration the need for adequate care for both critical (T1) and moderate (T2) trauma patients. The objective of this study was to verify the validity of these scales in nine hospitals of the Milano (Italy) metropolitan area that prepared for a possible MCI during EXPO 2015

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers

Neurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon. Neurons distribute signaling receptors to distal axons to receive extracellular information. Focusing on the neurotrophic receptor TrkB, Zahavi et al. elucidate an intracellular trafficking pathway that enables neurons to drive TrkB from its site of synthesis at the cell body, via secretory transport carriers, into the distal axon

Identification and characterization of Crumbs polarity complex proteins in Caenorhabditis elegans

Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting the formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including the scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here, we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1 and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on the physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues and, therefore, not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved