Exploring cannabidiol effects on inflammatory markers in individuals with cocaine use disorder: a randomized controlled trial.

Cocaine use disorder (CUD) is a major public health issue associated with physical, social, and psychological problems. Excessive and repeated cocaine use induces oxidative stress leading to a systemic inflammatory response. Cannabidiol (CBD) has gained substantial interest for its anti-inflammatory properties, safety, and tolerability profile. However, CBD anti-inflammatory properties have yet to be confirmed in humans. This exploratory study is based on a single-site randomized controlled trial that enrolled participants with CUD between 18 and 65 years, randomized (1:1) to daily receive either CBD (800 mg) or placebo for 92 days. The trial was divided into a 10-day detoxification (phase I) followed by a 12-week outpatient follow-up (phase II). Blood samples were collected from 48 participants at baseline, day 8, week 4, and week 12 and were analyzed to determine monocytes and lymphocytes phenotypes, and concentrations of various inflammatory markers such as cytokines. We used generalized estimating equations to detect group differences. Participants treated with CBD had lower levels of interleukin-6 (p = 0.017), vascular endothelial growth factor (p = 0.032), intermediate monocytes CD14+CD16+ (p = 0.024), and natural killer CD56negCD16hi (p = 0.000) compared with participants receiving placebo. CD25+CD4+T cells were higher in the CBD group (p = 0.007). No significant group difference was observed for B lymphocytes. This study suggests that CBD may exert anti-inflammatory effects in individuals with CUD.

Founder BRCA1/BRCA2/PALB2 pathogenic variants in French-Canadian breast cancer cases and controls.

Inherited germline pathogenic variants are responsible for ~5% of breast cancer globally. Through rapid expansion and isolation since immigration in the early 17th century, French Canadians are a relatively genetically homogenous founder population and therefore represent a unique demographic for genetic contributions to disease. To date, twenty variants in BRCA1, BRCA2, and PALB2 that predispose families to breast and ovarian cancer have been identified as recurring in the French-Canadian founder population. Our objective was to evaluate the clinical efficacy and validity of targeted genetic testing for these variants in Montreal French Canadians. A total of 555 breast cancer cases unselected for family history or age of diagnosis were genotyped, along with 1940 controls without a personal or family history of cancer. A Sequenom genotyping assay identified a pathogenic variant in 0.2% (5 of 1940) of cancer-free controls, and 3.8% (21/555) of breast cancer cases. Almost 10% (12/113) of early onset cases were heterozygous for founder BRCA1 or BRCA2 pathogenic variant. Of twenty variants tested, only seven were identified in this study. The option of providing this test as population-based screening is discussed.

Experienced pediatric nurses' perceptions of work-related stressors on general medical and surgical units: A qualitative study.

BACKGROUND: Experienced pediatric nurses caring for increasingly sick and vulnerable children on medical and surgical units may be at particular risk for work-related stress. In view of their positive impact on quality of care, and the fact that they are particularly difficult to retain, it is imperative to understand the work-related stressors these nurses encounter in order to develop effective organizational interventions to minimize stressors and promote retention. OBJECTIVE: To explore experienced pediatric nurses' perceptions of work-related stressors in medical and surgical units. DESIGN: Qualitative descriptive design with semi-structured interviews. SETTING: Medical and surgical units at a quaternary care pediatric hospital in Montreal, Canada. PARTICIPANTS: Nurses recognized as experienced by the nursing leadership team as reflected by having been 'in charge' of the unit, or having trained junior staff, and who had been practicing full-time for three years or more on a general medical or surgical pediatric unit were eligible to participate. Purposive sampling was used, and nurses recruited until data saturation was reached (n=12). There were no refusals to participate. METHODS: Semi-structured interviews were conducted between August and December 2013. RESULTS: Nurses described a strong sense of responsibility for providing excellent patient care, and identified stressor that negatively impacted their ability to do so. Stressors are reflected in three themes: (1) "The kids are getting sicker and sicker": Difficulty ensuring excellent patient care to an increasingly vulnerable population, (2) Feeling powerless to provide quality care, and (3) Being a "Jack-of-all-trades": Struggling with competing demands. CONCLUSION: Experienced pediatric nurses felt powerless to provide quality care to an increasingly acute and vulnerable population. Dealing with multiple and diverse responsibilities, and limited resources and support, were important stressors. Nurse Managers and educators could mitigate stressors and improve retention of experienced pediatric nurses by offering targeted continuing education to those newly responsible for additional roles, and building supportive working environments that encourage collaboration and empower experienced nurses.

PAPC mediates self/non-self-distinction during Snail1-dependent tissue separation.

Cleft-like boundaries represent a type of cell sorting boundary characterized by the presence of a physical gap between tissues. We studied the cleft-like ectoderm-mesoderm boundary in Xenopus laevis and zebrafish gastrulae. We identified the transcription factor Snail1 as being essential for tissue separation, showed that its expression in the mesoderm depends on noncanonical Wnt signaling, and demonstrated that it enables paraxial protocadherin (PAPC) to promote tissue separation through two novel functions. First, PAPC attenuates planar cell polarity signaling at the ectoderm-mesoderm boundary to lower cell adhesion and facilitate cleft formation. Second, PAPC controls formation of a distinct type of adhesive contact between mesoderm and ectoderm cells that shows properties of a cleft-like boundary at the single-cell level. It consists of short stretches of adherens junction-like contacts inserted between intermediate-sized contacts and large intercellular gaps. These roles of PAPC constitute a self/non-self-recognition mechanism that determines the site of boundary formation at the interface between PAPC-expressing and -nonexpressing cells.

Dynamin-dependent maintenance of epithelial integrity is essential for zebrafish epiboly.

Epiboly, the thinning and spreading of one tissue over another, is a widely employed morphogenetic movement that is essential for the development of many organisms. In the zebrafish embryo, epiboly describes the coordinated vegetal movement of the deep cells, enveloping layer (EVL) and yolk syncytial layer (YSL) to engulf the yolk cell. Recently, we showed that the large GTPase Dynamin plays a fundamental role in epiboly in the early zebrafish embryo. Because Dynamin plays a well-described role in vesicle scission during endocytosis, we predicted that Dynamin might regulate epiboly through participating in bulk removal of the yolk cell membrane ahead of the advancing margin, a proposed part of the epiboly motor. Unexpectedly, we found that Dynamin function was dispensable in the yolk cell and instead, it was required to maintain the epithelial integrity of the EVL during epiboly. Here, we present a model describing the maintenance of EVL integrity, which is required for the proper generation and transmission of tension during epiboly. Furthermore, we discuss the role of Dynamin-mediated regulation of ezrin-radixin-moesin (ERM) family proteins in the maintenance of epithelial integrity.

Zebrafish Dynamin is required for maintenance of enveloping layer integrity and the progression of epiboly.

Epiboly, the first morphogenetic cell movement that occurs in the zebrafish embryo, is the process by which the blastoderm thins and spreads to engulf the yolk cell. This process requires the concerted actions of the deep cells, the enveloping layer (EVL) and the extra-embryonic yolk syncytial layer (YSL). The EVL is mechanically coupled to the YSL which acts as an epiboly motor, generating the force necessary to draw the blastoderm towards the vegetal pole though actomyosin flow and contraction of the actomyosin ring. However, it has been proposed that the endocytic removal of yolk cell membrane just ahead of the advancing blastoderm may also play a role. To assess the contribution of yolk cell endocytosis in driving epiboly movements, we used a combination of drug- and dominant-negative-based approaches to inhibit Dynamin, a large GTPase with a well-characterized role in vesicle scission. We show that Dynamin-dependent endocytosis in the yolk cell is dispensable for epiboly of the blastoderm. However, global inhibition of Dynamin function revealed that Dynamin plays a fundamental role within the blastoderm during epiboly, where it maintains epithelial integrity and the transmission of tension across the EVL. The epithelial defects were associated with disrupted tight junctions and a striking reduction of cortically localized phosphorylated ezrin/radixin/moesin (P-ERM), key regulators of epithelial integrity in other systems. Furthermore, we show that Dynamin maintains EVL and promotes epiboly progression by antagonizing Rho A activity.

MFG-E8 released by apoptotic endothelial cells triggers anti-inflammatory macrophage reprogramming.

Apoptotic endothelial cells are an important component of the "response to injury" process. Several atherosclerosis risk factors such as hyperglycemia and oxidized low-density lipoproteins, and immune injuries, such as antibodies and complement, induce endothelial cell apoptosis. While endothelial cell apoptosis is known to affect neighboring vascular wall cell biology, its consequences on macrophage reprogramming are ill defined. In this study, we report that apoptosis of human and mouse endothelial cells triggers the release of milk fat globule-epidermal growth factor 8 (MFG-E8) and reprograms macrophages into an anti-inflammatory cells. We demonstrated that MFG-E8 is released by apoptotic endothelial cells in a caspase-3-dependent manner. When macrophages were exposed to conditioned media from serum-starved apoptotic endothelial cells, they adopt a high anti-inflammatory, low pro-inflammatory cytokine/chemokine secreting phenotype that is lost if MFG-E8 is absent from the media. Macrophage treatment with recombinant MFG-E8 recapitulates the effect of conditioned media. Finally, we showed that MFG-E8-mediated reprogramming of macrophages occurs through increased phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Taken together, our study suggests a key role of MFG-E8 release from apoptotic endothelial cells in macrophage reprogramming and demonstrates the importance of the apoptotic microenvironment in anti-inflammatory macrophage responses.

Zebrafish epiboly: mechanics and mechanisms.

Gastrulation involves of a series of coordinated cell movements to organize the germ layers and establish the major body axes of the embryo. One gastrulation movement is epiboly, which involves the thinning and spreading of a multilayered cell sheet. Epiboly plays a prominent role in zebrafish gastrulation and studies of zebrafish epiboly have provided insights into basic cellular properties and mechanisms of morphogenesis that are widely used in animal development. Although considerable progress has been made in identifying molecules that are required for epiboly, we still understand very little about how these factors cooperate to drive the process. Here, we review work on the molecular and cellular basis of zebrafish epiboly in order to identify unifying themes and to highlight some of the current open questions.

Caspase-3 activation triggers extracellular cathepsin L release and endorepellin proteolysis.

Proteolysis of extracellular matrix components and the production of cryptic bioactive factors play key roles in vascular remodeling. We showed previously that extracellular matrix proteolysis is triggered by the apoptosis of endothelial cells (EC), resulting in the release of an anti-apoptotic C-terminal fragment of endorepellin (LG3). Here, we characterize the endorepellin-cleaving proteases released by apoptotic EC using a multifaceted proteomics strategy. Cathepsin L (CathL), a cysteine protease known to be associated with cardiovascular disease progression in animal models and humans, was isolated from medium conditioned by apoptotic EC. CathL cleaved recombinant endorepellin in vitro, leading to LG3 release. Inhibition of CathL activity in EC exposed to pro-apoptotic stimuli prevented LG3 release without modulating the development of apoptosis in EC. Inhibition of caspase-3 activation in EC with the biochemical inhibitor DEVD-fluoromethyl ketone or small interfering RNAs concomitantly prevented CathL release by EC, LG3 production, and the development of paracrine anti-apoptotic activity. These data demonstrate that caspase-3 activation is a novel pathway of importance for triggering extracellular CathL release and the cleavage of extracellular matrix components.

Characterization and comparative expression of zebrafish calpain system genes during early development.

The classic calpain system has been implicated in regulating a variety of cellular processes including cell adhesion, migration, and intracellular signaling; however, little is known regarding the function of this system in vivo. Two heterodimeric Ca(2+)-dependent cysteine proteases, mu-calpain (CAPN1) and m-calpain (CAPN2), and the endogenous inhibitor calpastatin (CAST) comprise the classic/ubiquitous calpain system in mammals. Recently, knockout of two murine classic calpain genes, Capn2 and Capn4/Capns1, revealed that components of the classic system are indispensable for preimplantation development. We identified four classic calpain catalytic subunit genes (capn1a, 1b, 2a, 2b), two regulatory subunit genes (capns1a, 1b), and calpastatin (cast) from the zebrafish. Our data suggest that the components of the classic mammalian system are both conserved and expanded in the teleost lineage. In contrast to the classic/ubiquitous mammalian system, zebrafish calpain system genes acquire unique, tissue-specific patterns of expression within the first 2 days of development.

Melanosomal targeting sequences from gp100 are essential for MHC class II-restricted endogenous epitope presentation and mobilization to endosomal compartments.

CD4+ T lymphocytes play an important role in CD8+ T cell-mediated responses against tumors. Considering that approximately 20% of melanomas express MHC class II, it is plausible that concomitant presentation by MHC class I and class II shapes positive (helper T cells) or negative (regulatory T cells) antitumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we showed that gp100 putative NH2-terminal signal sequence and the last 70 residues in COOH terminus are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1+/HLA-DR+ endosomal/MIIC. Gp100 targeting sequences were characterized by deleting different sections in the COOH terminus (last 70 residues). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in COOH terminus resulted in decreased MHC class II presentation, without effects on class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100 targeting sequences to mobilize green fluorescent protein to endosomal compartments and to allow MHC class II and class I presentation of minimal endogenous epitopes. We conclude that these specific sequences are MIIC-targeting motifs, which could be included in expression cassettes for endogenously expressed tumor or viral antigens for MHC class II and class I presentation and optimize in vivo T-cell responses or as an in vitro tool for characterization of new MHC class II epitopes.