ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.

Machine learning workflows identify a microRNA signature of insulin transcription in human tissues.

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic ß-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription.

Laboratory and field evaluation of real-time and near real-time PM2.5 smoke monitors.

Increases in large wildfire frequency and intensity and a longer fire season in the western United States are resulting in a significant increase in air pollution, including concentrations of PM2.5 (particulate matter <2.5 µm in aerodynamic diameter) that pose significant health risks to nearby communities. During wildfires, government agencies monitor PM2.5 mass concentrations providing information and actions needed to protect affected communities; this requires continuously measuring instruments. This study assessed the performance of seven candidate instruments: (1) Met One Environmental beta attenuation monitor (EBAM), (2) Met One ES model 642 (ES642), (3) Grimm Environmental Dust Monitor 164 (EDM), (4) Thermo ADR 1500 (ADR), (5) TSI DRX model 8543 (DRX), (6) Dylos 1700 (Dylos), and (7) Purple Air II (PA-II) in comparison with a BAM 1020 (BAM) reference instrument. With the exception of the EBAM, all candidates use light scattering to determine PM2.5 mass concentrations. Our comparison study included environmental chamber and field components, with two of each candidate instrument operating next to the reference instrument. The chamber component involved 6 days of comparisons for biomass combustion emissions. The field component involved operating all instruments in an air monitoring station for 39.5 days with hourly average relative humidity (RH) ranging from 19% to 98%. Goals were to assess instrument precision and accuracy and effects of RH, elemental carbon (EC), and organic carbon (OC) concentrations. All replicate candidate instruments showed high hourly correlations (R2 ≥ 0.80) and higher daily average correlations (R2 ≥ 0.90), where all instruments correlated well (R2 ≥ 0.80) with the reference. The DRX and Purple Air overestimated PM2.5 mass concentrations by a factor of ~two. Differences between candidates and reference were more pronounced at higher PM2.5 concentrations. All optical instruments were affected by high RH and by the EC/OC ratio. Equations to convert candidate instruments data to FEM BAM type data are provided to enhance the usability of data from candidate instruments.Implications: This study tested the performance of seven candidate PM2.5 mass concentration measuring instruments in two settings - environmental chamber and field. The instruments were tested to determine their suitability for use during biomass combustion events and the effects of RH, PM mass concentrations, and concentrations of EC and OC on their performance. The accuracy and precision of each monitor and effect of RH, PM concentration, EC and OC concentrations are varied. The data show that most of these candidate instruments are suitable for measuring PM2.5 concentration during biomass combustions with a proper correction factor for each instrument type.

The RhoGAP protein ARHGAP18/SENEX localizes to microtubules and regulates their stability in endothelial cells.

RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that ARHGAP18 in endothelial cells is important in cell junctions. Here we show, using structured illumination microscopy (SIM), ground-state depletion (GSD), and total internal reflection fluorescence microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and histone deacetylase 6 but not by a GAP-mutant form of ARHGAP18. Depletion of ARHGAP18 resulted in a failure to secrete endothelin-1 and a reduction in neutrophil transmigration, both known to be microtubule dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilization, enhanced the plasma membrane-bound fraction of ARHGAP18. Thus, in endothelial cells, ARHGAP18 may act as a significant regulator of vascular homeostasis.

An Intervention to Improve the Comfort And Satisfaction of Nurses in the Telephone Triage of Child Maltreatment Calls.

Nurses are mandated reporters of actual or suspected child maltreatment or the threat thereof. The purpose of this quality improvement project was to determine the knowledge and comfort of nurses in telephone triage in pediatric clinics when dealing with suspected or actual child abuse calls. Nurses (N = 17) from three pediatric primary care clinics and one specialty care orthopedic clinic were surveyed. Based on results of the survey showing a lack of knowledge and adequate referral resources perceived by the nursing staff, resources and staff education were developed, along with a script for guiding maltreatment calls toward standardization of care. Following the intervention, nurses reported an increased comfort level when doing telephone triage for child maltreatment calls, an increase in knowledge of risk factors for county resources. Further, they reported a substantial shift in opinion about the need for a standardized script when responding to child maltreatment telephone calls. Nurses undertaking telephone triage of high-risk child maltreatment calls can improve their comfort and knowledge through a survey of their needs and directed education and resource development for the management of child maltreatment telephone triage.

A survey of wellness management strategies used by Canadian doctors of chiropractic.

OBJECTIVE: The purpose of this study was to investigate if Canadian doctors of chiropractic consider using wellness strategies after functional recovery in acute and chronic conditions. This study also attempted to determine if there is a difference in the use of wellness management strategies between broad and narrow scope practitioners. METHODS: Forty-one practicing, licensed chiropractors were recruited to complete an interview survey regarding 2 mock clinical case presentations. Interviews were recorded, and influential words or word pairings were identified. Investigators formulated criteria to divide practitioners into broad scope (mixers) and narrow scope (straights). Data were analyzed using Crawdad Analysis Software (version 1.2). RESULTS: All subjects indicated that they would provide information regarding public health and wellness strategies to a patient after functional resolution of the presenting chronic or acute complaints. The responses of broad scope (mixer) chiropractors appeared to be focused on the patient specifically, whereas narrow scope (straight) responses appeared to be more varied when analyzed for noun and noun-pair influence. CONCLUSION: This study of practicing, licensed Canadian chiropractors suggests that wellness strategies may be commonly considered in practice. All subjects in this study reported a number of strategies to educate patients regarding wellness after functional recovery of a complaint.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein.

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor.

Phosphorylation of cold shock domain/Y-box proteins by ERK2 and GSK3beta and repression of the human VEGF promoter.

The hypoxia responsive region (HRR) of the VEGF promoter plays a key role in regulating VEGF expression. We found that the cold shock domain (Y-box) repressor proteins, dbpA and dbpB/YB-1, bind distinct strands of the human VEGF HRR. We find both dbpA and dbpB are phosphorylated by ERK2 and GSK3beta in vitro, and the binding of dbpB to single-strand VEGF HRR DNA is regulated by this phosphorylation. These findings suggest the ERK/MAPK and PI3K pathways may regulate VEGF expression in part through regulating the action of these repressor proteins.

A multi-protein complex containing cold shock domain (Y-box) and polypyrimidine tract binding proteins forms on the vascular endothelial growth factor mRNA. Potential role in mRNA stabilization.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and post-transcriptional regulation plays a major role in VEGF expression. Both the 5'- and 3'-UTR are required for VEGF post-transcriptional regulation but factors binding to functional sequences within the 5'-UTR have not been fully characterized. We report here the identification of complexes, binding to the VEGFmRNA 5'- and 3'-UTR, that contain cold shock domain (CSD) and polypyrimidine tract binding (PTB) RNA binding proteins. Analysis of the CSD/PTB binding sites revealed a potential role in VEGF mRNA stability, in both noninduced and induced conditions, demonstrating a general stabilizing function. Such a stabilizing mechanism had not been reported previously for the VEGF gene. We further found that the CSD/PTB-containing complexes are large multiprotein complexes that are most likely preformed in solution and we demonstrate that PTB is associated with the VEGF mRNA in vivo. Complex formation between CSD proteins and PTB has not been reported previously. Analysis of the CSD/PTB RNA binding sites revealed a novel CSD protein RNA recognition site and also demonstrated that CSD proteins may direct the binding of CSD/PTB complexes. We found the same complexes binding to an RNA-stabilizing element of another growth factor gene, suggesting a broader functional role for the CSD/PTB complexes. Finally, as the VEGF gene is also regulated at the transcriptional level by CSD proteins, we propose a combined transcriptional/post-transcriptional role for these proteins in VEGF and other growth factor gene regulation.

A novel mechanism of repression of the vascular endothelial growth factor promoter, by single strand DNA binding cold shock domain (Y-box) proteins in normoxic fibroblasts.

Overexpression of vascular endothelial growth factor (VEGF) is implicated in a number of diseases. It is therefore critical that mechanisms exist to strictly regulate VEGF expression. A hypoxia-responsive (HR) region of the VEGF promoter which binds the HIF-1 transcription factor is a target for many signals that up-regulate VEGF transcription. Repressors targeting the HIF-1 transcription factor have been identified but no repressors directly binding the HR promoter region had been reported. We now report a novel mechanism of repression of the VEGF HR region involving DNA binding. We find that single strand DNA-specific cold shock domain (CSD or Y-box) proteins repress the HR region via a binding site downstream of the HIF-1 site. The repressor site is functional in unstimulated, normoxic fibroblasts and represents a novel means to prevent expression of VEGF in the absence of appropriate stimuli. We characterized complexes forming on the VEGF repressor site and identified a previously unreported nuclear CSD protein complex containing dbpA. Nuclear dbpA appears to bind as a dimer and we determined a means by which nuclear CSD proteins may enter double strand DNA to bind to their single strand sites to bring about repression of the VEGF HR region.