The functional organization of axonal mRNA transport and translation.

Axons extend for tremendously long distances from the neuronal soma and make use of localized mRNA translation to rapidly respond to different extracellular stimuli and physiological states. The locally synthesized proteins support many different functions in both developing and mature axons, raising questions about the mechanisms by which local translation is organized to ensure the appropriate responses to specific stimuli. Publications over the past few years have uncovered new mechanisms for regulating the axonal transport and localized translation of mRNAs, with several of these pathways converging on the regulation of cohorts of functionally related mRNAs - known as RNA regulons - that drive axon growth, axon guidance, injury responses, axon survival and even axonal mitochondrial function. Recent advances point to these different regulatory pathways as organizing platforms that allow the axon's proteome to be modulated to meet its physiological needs.

Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs.

Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3'UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.

Axonal localization of neuritin/CPG15 mRNA is limited by competition for HuD binding.

HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.

The ubiquitin ligase ITCH coordinates small intestinal epithelial homeostasis by modulating cell proliferation, differentiation, and migration.

Maintenance of the intestinal mucosa is driven by local signals that coordinate epithelial proliferation, differentiation, and turnover in order to separate antigenic luminal contents from the host's immune system. Breaches in this barrier promote gastrointestinal pathologies ranging from inflammatory bowel disease to cancer. The ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders, particularly in the colon. However, the small intestine appears to be spared from this pathology. Here we explored the physiological mechanism that underlies the preservation of mucosal homeostasis in the small intestine in mice lacking ITCH (Itcha18H/a18H). Histological analysis of the small intestines from young adult mice revealed architectural changes in animals deficient for ITCH, including villus blunting with cell crowding, crypt expansion, and thickening of the muscularis propria relative to age-matched mice sufficient for ITCH. These differences were more prominent in the distal part of the small intestine and were not dependent upon lymphoid cells. Underlying the observed changes in the epithelium were expansion of the Ki67+ proliferating transit amplifying progenitor population and increased numbers of terminally differentiated mucus-secreting goblet and anti-microbial producing Paneth cells, which are both important in controlling local inflammation in the small intestine and are known to be dysregulated in inflammatory bowel disease. Homeostasis in the small intestine of Itcha18H/a18H animals was maintained by increased cell turnover, including accelerated migration of epithelial cells along the crypt-villus axis and increased apoptosis of epithelial cells at the crypt-villus junction. Consistent with this enhanced turnover, Itcha18H/a18H mice carrying the Min mutation (Itcha18H/a18H; ApcMin/+) displayed a 76% reduction in tumor burden as compared to ApcMin/+ littermates with normal levels of ITCH. These findings highlight the role of ITCH as an important modulator of intestinal epithelial homeostasis.

Comparison of the Trends in Risk Factors and Management of Severe Postpartum Hemorrhage Years 2000-2004 Versus 2005-2008.

OBJECTIVE: To compare trends in the etiology and management of severe postpartum hemorrhage (PPH) during 2 time periods: 2000-2004 (Period 1) versus 2005-2008 (Period 2). STUDY DESIGN: Medical records with a diagnosis of PPH were identified by ICD-9 codes for immediate, third-stage, delayed, and secondary. PPH and post- partum coagulation defect. Subjects having a PPH within 24 hours of delivery who also received blood component therapy (defined as severe PPH) during Period 1 were compared with those from Period 2. RESULTS: There were 109 and 119 cases identified from Periods 1 and 2, respectively. Uterine atony was the most common cause of severe PPH during both time periods. In the second time period women with severe PPH had a lower mean hematocrit (p<0.05), a greater mean BMI (p<0.05), and more induced labor (p<0.01) as compared to the first time period. A greater proportion of the women in the second time period received misoprostol (p<0.0001) and platelets (p<0.05). The proportions of other therapies and surgical interventions remained unchanged, as did the ultimate outcomes. CONCLUSION: At a single large institution over the course of a 9-year period the management of severe PPH changed to include a greater utilization of misoprostol and platelet therapy.

Cardiomyocyte-specific overexpression of the ubiquitin ligase Wwp1 contributes to reduction in Connexin 43 and arrhythmogenesis.

Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a ß-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.

An epidemiologic study comparing cancer- and noncancer-associated venous thromboembolism in a racially diverse Southeastern United States county.

Background: Cancer-associated venous thromboembolism (CA-VTE) represents a major cause of morbidity and mortality in patients with cancer. Despite poor outcomes, there is an ongoing knowledge gap in epidemiologic data related to this association. Objectives: To compare venous thromboembolism (VTE) characteristics, risk factors, and outcomes between patients with and without active cancer in a racially diverse population. Methods: Our surveillance project occurred at the 3 hospitals in Durham County, North Carolina, from April 2012 through March 2014. Electronic and manual methods were used to identify unique Durham County residents with VTE. Results: We identified 987 patients with VTE during the surveillance period. Of these, 189 patients had active cancer at the time of their VTE event. Patients with CA-VTE were older (median age: 69 years vs 60 years, P < .0001) and had a lower body mass index (median body mass index: 26.0 kg/m2 vs 28.4 kg/m2, P = .0001) than noncancer patients. The most common cancers in our cohort were gastrointestinal, breast, genitourinary, and lung. The proportion of VTE cases with pulmonary embolism (PE) was greater in the cancer cohort compared with that in the noncancer cohort (58.2% vs 44.0%, P = .0004). Overall survival was lower in the CA-VTE group than in patients without cancer (P < .0001). Black patients with CA-VTE had lower proportion of PE (52.3% vs 67.1%, P = .05) but had decreased survival (P < .0003) in comparison with White patients. Conclusion: Future studies may be needed to continue to evaluate local and national VTE data to improve VTE prevention strategies and CA-VTE outcomes.

Racial differences in venous thromboembolism: A surveillance program in Durham County, North Carolina.

Background: Venous thromboembolism (VTE) affects approximately 1-2 individuals per 1000 annually and is associated with an increased risk for pulmonary hypertension, postthrombotic syndrome, and recurrent VTE. Objective: To determine risk factors, incidence, treatments, and outcomes of VTE through a 2-year surveillance program initiated in Durham County, North Carolina (population approximately 280,000 at time of study). Patients/Methods: We performed a retrospective analysis of data actively collected from three hospitals in Durham County during the surveillance period. Results: A total of 987 patients were diagnosed with VTE, for an annual rate of 1.76 per 1000 individuals. Hospital-associated VTE occurred in 167 hospitalized patients (16.9%) and 271 outpatients who were hospitalized within 90 days of diagnosis (27.5%). Annual incidence was 1.98 per 1000 Black individuals compared to 1.25 per 1000 White individuals (p < 0.0001), and Black individuals with VTE were younger than White individuals (p < 0.0001). Common risk factors included active cancer, prolonged immobility, and obesity, and approximately half were still taking anticoagulant therapy 1 year later. A total of 224 patients died by 1 year (28.5% of patients for whom outcomes could be confirmed), and Black patients were more likely to have recurrent VTE than White patients during the first 6 months following initial presentation (9.4% vs. 4.1%, p = 0.01). Conclusions: Ongoing surveillance provides an effective strategy to identify patients with VTE and monitor treatment and outcomes. We demonstrated that hospital-associated VTE continues to be a major contributor to the burden of VTE and confirmed the higher incidence of VTE in Black compared to White individuals.

MicroRNAs 21 and 199a-3p Regulate Axon Growth Potential through Modulation of Pten and mTor mRNAs.

Increased mTOR activity has been shown to enhance regeneration of injured axons by increasing neuronal protein synthesis, while PTEN signaling can block mTOR activity to attenuate protein synthesis. MicroRNAs (miRs) have been implicated in regulation of PTEN and mTOR expression, and previous work in spinal cord showed an increase in miR-199a-3p after spinal cord injury (SCI) and increase in miR-21 in SCI animals that had undergone exercise. Pten mRNA is a target for miR-21 and miR-199a-3p is predicted to target mTor mRNA. Here, we show that miR-21 and miR-199a-3p are expressed in adult dorsal root ganglion (DRG) neurons, and we used culture preparations to test functions of the rat miRs in adult DRG and embryonic cortical neurons. miR-21 increases and miR-199a-3p decreases in DRG neurons after in vivo axotomy. In both the adult DRG and embryonic cortical neurons, miR-21 promotes and miR-199a-3p attenuates neurite growth. miR-21 directly bound to Pten mRNA and miR-21 overexpression decreased Pten mRNA levels. Conversely, miR-199a-3p directly bound to mTor mRNA and miR-199a-3p overexpression decreased mTor mRNA levels. Overexpressing miR-21 increased both overall and intra-axonal protein synthesis in cultured DRGs, while miR-199a-3p overexpression decreased this protein synthesis. The axon growth phenotypes seen with miR-21 and miR-199a-3p overexpression were reversed by co-transfecting PTEN and mTOR cDNA expression constructs with the predicted 3' untranslated region (UTR) miR target sequences deleted. Taken together, these studies indicate that injury-induced alterations in miR-21 and miR-199a-3p expression can alter axon growth capacity by changing overall and intra-axonal protein synthesis through regulation of the PTEN/mTOR pathway.

A Ca2+-Dependent Switch Activates Axonal Casein Kinase 2α Translation and Drives G3BP1 Granule Disassembly for Axon Regeneration.

The main limitation on axon regeneration in the peripheral nervous system (PNS) is the slow rate of regrowth. We recently reported that nerve regeneration can be accelerated by axonal G3BP1 granule disassembly, releasing axonal mRNAs for local translation to support axon growth. Here, we show that G3BP1 phosphorylation by casein kinase 2α (CK2α) triggers G3BP1 granule disassembly in injured axons. CK2α activity is temporally and spatially regulated by local translation of Csnk2a1 mRNA in axons after injury, but this requires local translation of mTor mRNA and buffering of the elevated axonal Ca2+ that occurs after axotomy. CK2α's appearance in axons after PNS nerve injury correlates with disassembly of axonal G3BP1 granules as well as increased phospho-G3BP1 and axon growth, although depletion of Csnk2a1 mRNA from PNS axons decreases regeneration and increases G3BP1 granules. Phosphomimetic G3BP1 shows remarkably decreased RNA binding in dorsal root ganglion (DRG) neurons compared with wild-type and non-phosphorylatable G3BP1; combined with other studies, this suggests that CK2α-dependent G3BP1 phosphorylation on Ser 149 after axotomy releases axonal mRNAs for translation. Translation of axonal mRNAs encoding some injury-associated proteins is known to be increased with Ca2+ elevations, and using a dual fluorescence recovery after photobleaching (FRAP) reporter assay for axonal translation, we see that translational specificity switches from injury-associated protein mRNA translation to CK2α translation with endoplasmic reticulum (ER) Ca2+ release versus cytoplasmic Ca2+ chelation. Our results point to axoplasmic Ca2+ concentrations as a determinant for the temporal specificity of sequential translational activation of different axonal mRNAs as severed axons transition from injury to regenerative growth.

Design and Implementation of a Comprehensive Surveillance System for Venous Thromboembolism in a Defined Region Using Electronic and Manual Approaches.

BACKGROUND: Systematic surveillance for venous thromboembolism (VTE) in the United States has been recommended by several organizations. Despite adoption of electronic medical records (EMRs) by most health care providers and facilities, however, systematic surveillance for VTE is not available. OBJECTIVES: This article develops a comprehensive, population-based surveillance strategy for VTE in a defined geographical region. METHODS: The primary surveillance strategy combined computerized searches of the EMR with a manual review of imaging data at the Duke University Health System in Durham County, North Carolina, United States. Different strategies of searching the EMR were explored. Consolidation of results with autopsy reports (nonsearchable in the EMR) and with results from the Durham Veterans' Administration Medical Center was performed to provide a comprehensive report of new VTE from the defined region over a 2-year timeframe. RESULTS: Monthly searches of the primary EMR missed a significant number of patients with new VTE who were identified by a separate manual search of radiology records, apparently related to delays in data entry and coding into the EMR. Comprehensive searches incorporating a location-restricted strategy were incomplete due to the assigned residence reflecting the current address and not the address at the time of event. The most comprehensive strategy omitted the geographic restriction step and identified all patients with VTE followed by manual review of individual records to remove incorrect entries (e.g., outside the surveillance time period or geographic location; no evidence for VTE). Consolidation of results from the EMR searches with results from autopsy reports and the separate facility identified additional patients not diagnosed within the Duke system. CONCLUSION: We identified several challenges with implementing a comprehensive VTE surveillance program that could limit accuracy of the results. Improved electronic strategies are needed to cross-reference patients across multiple health systems and to minimize the need for manual review and confirmation of results.

Postpartum wound and bleeding complications in women who received peripartum anticoagulation.

INTRODUCTION: The objective of this study was to compare wound and bleeding complications between women who received anticoagulation after cesarean delivery due to history of prior venous thromboembolic disease, arterial disease, or being a thrombophilia carrier with adverse pregnancy outcome, to women not receiving anticoagulation. METHODS: Women in the Duke Thrombosis Center Registry who underwent cesarean delivery during 2003-2011 and received postpartum anticoagulation (anticoagulation group, n=77), were compared with a subset of women who delivered during the same time period, but did not receive anticoagulation (no anticoagulation group, n=77). The no anticoagulation group comprised women who were matched to the anticoagulation group by age, body mass index, type of cesarean (no labor vs. labor), and date of delivery. Bleeding and wound complications were compared between the two groups. A multivariable logistic regression model was constructed to determine if anticoagulation was an independent predictor of wound complication. RESULTS: Women who received anticoagulation during pregnancy had a greater incidence of wound complications compared to those who did not (30% vs. 8%, p<0.001). Using multivariable logistic regression, while controlling for race, diabetes, chorioamnionitis, and aspirin use, anticoagulation predicted the development of any wound complication (OR 5.8, 95% CI 2.2, 17.6), but there were no differences in the mean estimated blood loss at delivery (782 vs. 778 ml, p=0.91), change in postpartum hematocrit (5.4 vs. 5.2%, p=0.772), or percent of women receiving blood products (6.5 vs. 1.3%, p=0.209) between the two groups. CONCLUSIONS: Anticoagulation following cesarean delivery is associated with an increased risk of post-cesarean wound complications, but not other postpartum bleeding complications.

Direct-to-patient expert system and home INR monitoring improves control of oral anticoagulation.

BACKGROUND AND OBJECTIVE: Internet-based disease management programs have the potential to improve patient care. The objective of this study was to determine whether an interactive, internet-based system enabling supervised, patient self-management of oral anticoagulant therapy provided management comparable to an established anticoagulation clinic. PATIENTS/METHODS: Sixty patients receiving chronic oral anticoagulant therapy who had access to the internet and a printer, were enrolled into this prospective, single-group, before-after study from a single clinic and managed between March 2002 and January 2003. Patients learned how to use a home prothrombin time monitor and how to access the system through the internet. Patients used the system for six months, with daily review by the supervising physician. The primary outcome variable was the difference in time in therapeutic range prior to and following introduction of internet-supervised patient self-management. RESULTS: The mean time in therapeutic range increased from 63% in the anticoagulation clinic (control period) to 74.4% during internet-supervised patient self-management (study period). The mean difference score between control and study periods was 11.4% (P = 0.004, 95% confidence interval 5.5-17.3%). There were no hemorrhagic or thromboembolic complications. CONCLUSIONS: This novel approach of internet-supervised patient self-management improved time in therapeutic range compared to an anticoagulation clinic. This is the first demonstration of an internet-based expert system enabling remote and effective management of patients on oral anticoagulants. Expert systems may be applicable for management of other chronic diseases.

Development of a deer mouse whole-genome radiation hybrid panel and comparative mapping of Mus chromosome 11 loci.

A 5000-rad whole-genome radiation hybrid cell panel (BW5000) was developed for mapping the deer mouse (Peromyscus maniculatus bairdii) genome. The panel consists of 103 cell lines and has an estimated marker retention frequency of 63.9% (range, 28%-88%) based on PCR typing of 30 Type I (coding gene) and 25 Type II (microsatellite) markers. Using the composite Mus map, Type I markers were selected from six Mus chromosomes, 22 of which are on Mus Chr 11. Fifteen of the Mus Chr 11 markers were simultaneously mapped on an interspecific (P. maniculatus x P. polionotus) backcross panel to test the utility of the radiation hybrid panel, create a framework map, and help establish gene order. The radiation hybrids have effectively detected linkage in the deer mouse genome between markers as far apart as 6.7 cM and resolved markers that are, in the Mus genome, as close as 0.2 Mb. Combined results from both panels have indicated a high degree of gene order conservation of the telomeric 64 cM of Mus Chr 11 in the deer mouse genome. The remaining centromeric portion also shows gene order conservation with the deer mouse but as a separate linkage group. This indicates a translocation of that portion of Mus Chr 11 in P. maniculatus and is consistent with rearrangement breakpoints observed between Mus and other mammalian genomes, including rat and human. Furthermore, this separate linkage group is likely to reside in a chromosomal region of inversion polymorphism between P. maniculatus and P. polionotus.

Use of a new platelet function analyzer to detect von Willebrand disease in women with menorrhagia.

OBJECTIVE: The purpose of this study was to assess the usefulness of a new platelet function analyzer in the detection of von Willebrand disease and platelet dysfunction in women with menorrhagia. STUDY DESIGN: Women with menorrhagia and control subjects were tested with a platelet function analyzer. If the results were abnormal, further testing was performed for possible von Willebrand disease or platelet dysfunction. Results were compared. RESULTS: Of the 108 women with menorrhagia, 28 had an abnormal platelet function analysis results: 7 results were suggestive of von Willebrand disease; 17 results were suggestive of platelet dysfunction, and 4 results were inconclusive. Of the 100 control subjects, 18 subjects had an abnormal platelet function analysis result: 2 results were suggestive of von Willebrand disease; 12 results were suggestive of platelet dysfunction, and 4 results were inconclusive. The prevalence of suspected von Willebrand disease was 6% among women with menorrhagia and 2% among control subjects. Among white women, the prevalence was 10% compared with 1% among control subjects. CONCLUSION: We demonstrated that the platelet function analyzer could be used to detect von Willebrand disease in women with menorrhagia.

Antiphospholipid antibodies after surgical exposure to topical bovine thrombin.

Exposure to topical bovine thrombin during surgery frequently results in the development of antibodies to multiple protein and carbohydrate antigens. We investigated the frequency of increased levels of antibodies to cardiolipin and beta(2)-glycoprotein I (beta(2)-GPI) in two groups of patients, one exposed to bovine thrombin during cardiovascular surgery (n = 151) and a "control" group undergoing cardiovascular surgery but without exposure to bovine thrombin (n = 11). Anticardiolipin antibody levels were increased before surgery in 10 of the 151 patients exposed to topical thrombin (6.6%). Four to 8 weeks after surgery, 84 patients (55.6%) had increased anticardiolipin antibody levels (P <.0001). In the control group, an increased anticardiolipin antibody level was present in a single patient before and after surgery (9%). Increased levels of antibodies to bovine and human beta(2)-GPI were also observed after surgery in the patients exposed to topical thrombin (37.7% and 38.2%, respectively). Increased anticardiolipin levels correlated with higher levels of antibody to bovine, but not human, beta(2)-GPI. In addition, increased levels of anticardiolipin antibody were associated with higher levels of antibodies to bovine factor V and prothrombin, as well as human factor V. Antibody binding on an enzyme-linked immunosorbent assay conducted to detect anticardiolipin antibody was dependent on the presence of anionic phospholipid, indicating that binding was not linked to the fetal bovine serum in the blocking buffer alone. Seven of 8 patients with delayed thromboembolic complications had increased anticardiolipin IgG antibody levels after surgery, but this association was not statistically significant. Nevertheless, our findings support the recommendation that the clinical safety of these commonly used hemostatic agents should be reassessed.