Characterization of the roles of the Nck interacting kinase MIG-15 and the Rac GTPases in neuronal migration in Caenorhabditis elegans.

Neuronal migration is essential to the formation of the central nervous system in vertebrates. In Caenorhabditis elegans, a screen was performed previously to identify mutations that affected the migration of the Q neuroblast descendants. One of the mutants isolated from this screen was mig-15. MIG-15, a Nck Interacting Kinase (NIK), is homologous to proteins found in a wide variety of organisms, including Drosophila, mice, and humans, in which NIK kinases have been implicated in cell migration. Interestingly, multiple components of the canonical Wnt signaling pathway had already been found to control the Q cell descendant migrations. Additionally, the MIG-15 homolog in Drosophila, Misshapen had also been found to work with Wnt signaling components in the non-canonical planar cell polarity pathway. To determine how MIG-15 was working to control the migrations of the Q cell descendants, a characterization of the Q neuroblast migration defects was performed. mig-15 mutants were found to affect the Q neuroblasts, along with their descendants as previously described. I carried this work further and found that MIG-15 is required for extension of lamellipodial protrusions, maintenance of the initial polarization directing these initial protrusions, and migration of the Q neuroblasts. Since the Wnt signaling pathway had been implicated in Q cell descendant migration as well, several Wnt signaling mutants were also examined in the Q neuroblasts. This analysis determined that for the Wnt signaling mutants that were observed, there was no effect on early Q neuroblast protrusion extension or migration. Therefore, MIG-15 does not appear to be acting with the Wnt signaling pathway to control Q neuroblast migration. Subsequently, the Q cell descendant migrations of the AQR and PQR neurons were also examined for both mig-15 and Wnt signaling mutants. Double mutants of mig-15 with Wnt signaling mutants resembled mig-15 mutants alone, further suggesting that MIG-15 is not working with the Wnt signaling pathway to control the Q neuroblast lineage migrations. In attempt to elucidate how MIG-15 is controlling the migrations of the Q neuroblasts and descendants, a candidate gene approach was taken to determine other possible proteins that are required for Q neuroblast migration. The C-terminal region of MIG-15 had previously been found to bind to PAT-3, the beta-integrin homolog in C. elegans. Since available mutants in pat-3 are not viable, INA-1/alpha-integrin was examined for defects in Q neuroblast migration. This analysis found that, like MIG-15, INA-1 is required for the extension of polarized protrusions and migration of the Q neuroblasts. Though, INA-1 was not involved in maintenance of polarization as was MIG-15. Another molecule that was examined was ERM-1, the C. elegans homolog of the ezrin, radixin, and moesin (ERM) family of proteins. Previous studies have found that ERM proteins bind to and are phosphorylated by Nck interacting kinases in cell culture. These studies found that removal of ERM-1 from mig-15 mutants suppressed the migration defects seen for the QL neuroblasts in the mig-15 mutants alone. Together, these results suggest that MIG-15 could be acting with the integrins for proper polarization and with both ERM-1 and the integrins to direct migration of the Q neuroblasts and descendants. Previous studies had suggested that MIG-15 works upstream of the Rac GTPases MIG-2 and CED-10 in axon pathfinding. Additionally, these molecules had been found to act in parallel to control axon pathfinding. In order to determine if these Rac GTPases are also required for migration of the Q neuroblasts, single and double mutants of the Rac GTPases and several other molecules that are known to work with these molecules in other systems, including CDC-42 and UNC-73, were examined for their effects on the Q neuroblasts and their migrations. Singly, mutants of mig-2, ced-10, and cdc-42 did not cause strong defects in the ability to extend protrusions or the migrations of the Q neuroblasts. When double mutants were analyzed, strong defects in the polarization and migration of the Q neuroblasts were observed for the mig-2;ced-10 double mutants. Previous data has suggested that UNC-73 acts as a guanine nucleotide exchange factor (GEF) for MIG-2 and CED-10. unc-73 mutants displayed polarization and migration defects like the mig-2; ced-10 double mutant, but the defects were less severe in the unc-73 mutants, suggesting that there is another GEF that facilitates GTP exchange for MIG-2 and CED-10. Observations of a second GEF, PIX-1, suggest that PIX-1 is involved in the protrusion extension and migration of the Q neuroblasts, suggesting that PIX-1 might act as the other GEF for MIG-2 and CED-10. Further studies found that PIX-1 might be functioning in a linear pathway with CED-10 and in parallel to the UNC-73/MIG-2 signaling pathway. In summary, my work reveals the roles of the Rac GTPases and MIG-15/NIK kinase in the migrations of the Q neuroblasts in Caenorhabditis elegans, which provides insight into the mechanisms that drive neuronal migration during nervous system development

The Arp2/3 Complex, UNC-115/abLIM, and UNC-34/Enabled Regulate Axon Guidance and Growth Cone Filopodia Formation in Caenorhabditis Elegans

Background While many molecules involved in axon guidance have been identified, the cellular and molecular mechanisms by which these molecules regulate growth cone morphology during axon outgrowth remain to be elucidated. The actin cytoskeleton of the growth cone underlies the formation of lamellipodia and filopodia that control growth cone outgrowth and guidance. The role of the Arp2/3 complex in growth cone filopodia formation has been controversial, and other mechanisms of growth cone filopodia formation remain to be described. Results Here we show that mutations in genes encoding the Arp2/3 complex (arx genes) caused defects in axon guidance. Analysis of developing growth cones in vivo showed that arx mutants displayed defects in filopodia and reduced growth cone size. Time-lapse analysis of growth cones in living animals indicated that arx mutants affected the rate of growth cone filopodia formation but not filopodia stability or length. Two other actin modulatory proteins, UNC-115/abLIM and UNC-34/Enabled, that had been shown previously to affect axon guidance had overlapping roles with Arp2/3 in axon guidance and also affected the rate of filopodia initiation but not stability or length. Conclusion Our results indicate that the Arp2/3 complex is required cell-autonomously for axon guidance and growth cone filopodia initiation. Furthermore, they show that two other actin-binding proteins, UNC-115/abLIM and UNC-34/Enabled, also control growth cone filopodia formation, possibly in parallel to Arp2/3. These studies indicate that, in vivo, multiple actin modulatory pathways including the Arp2/3 complex contribute to growth cone filopodia formation during growth cone outgrowth

Effects of a high-dose 24-h infusion of tranexamic acid on death and thromboembolic events in patients with acute gastrointestinal bleeding (HALT-IT): an international randomised, double-blind, placebo-controlled trial

Background: Tranexamic acid reduces surgical bleeding and reduces death due to bleeding in patients with trauma. Meta-analyses of small trials show that tranexamic acid might decrease deaths from gastrointestinal bleeding. We aimed to assess the effects of tranexamic acid in patients with gastrointestinal bleeding. Methods: We did an international, multicentre, randomised, placebo-controlled trial in 164 hospitals in 15 countries. Patients were enrolled if the responsible clinician was uncertain whether to use tranexamic acid, were aged above the minimum age considered an adult in their country (either aged 16 years and older or aged 18 years and older), and had significant (defined as at risk of bleeding to death) upper or lower gastrointestinal bleeding. Patients were randomly assigned by selection of a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients received either a loading dose of 1 g tranexamic acid, which was added to 100 mL infusion bag of 0·9% sodium chloride and infused by slow intravenous injection over 10 min, followed by a maintenance dose of 3 g tranexamic acid added to 1 L of any isotonic intravenous solution and infused at 125 mg/h for 24 h, or placebo (sodium chloride 0·9%). Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was death due to bleeding within 5 days of randomisation; analysis excluded patients who received neither dose of the allocated treatment and those for whom outcome data on death were unavailable. This trial was registered with Current Controlled Trials, ISRCTN11225767, and ClinicalTrials.gov, NCT01658124. Findings: Between July 4, 2013, and June 21, 2019, we randomly allocated 12 009 patients to receive tranexamic acid (5994, 49·9%) or matching placebo (6015, 50·1%), of whom 11 952 (99·5%) received the first dose of the allocated treatment. Death due to bleeding within 5 days of randomisation occurred in 222 (4%) of 5956 patients in the tranexamic acid group and in 226 (4%) of 5981 patients in the placebo group (risk ratio [RR] 0·99, 95% CI 0·82–1·18). Arterial thromboembolic events (myocardial infarction or stroke) were similar in the tranexamic acid group and placebo group (42 [0·7%] of 5952 vs 46 [0·8%] of 5977; 0·92; 0·60 to 1·39). Venous thromboembolic events (deep vein thrombosis or pulmonary embolism) were higher in tranexamic acid group than in the placebo group (48 [0·8%] of 5952 vs 26 [0·4%] of 5977; RR 1·85; 95% CI 1·15 to 2·98). Interpretation: We found that tranexamic acid did not reduce death from gastrointestinal bleeding. On the basis of our results, tranexamic acid should not be used for the treatment of gastrointestinal bleeding outside the context of a randomised trial

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

Effects of a high-dose 24-h infusion of tranexamic acid on death and thromboembolic events in patients with acute gastrointestinal bleeding (HALT-IT): an international randomised, double-blind, placebo-controlled trial

BackgroundTranexamic acid reduces surgical bleeding and reduces death due to bleeding in patients with trauma. Meta-analyses of small trials show that tranexamic acid might decrease deaths from gastrointestinal bleeding. We aimed to assess the effects of tranexamic acid in patients with gastrointestinal bleeding.MethodsWe did an international, multicentre, randomised, placebo-controlled trial in 164 hospitals in 15 countries. Patients were enrolled if the responsible clinician was uncertain whether to use tranexamic acid, were aged above the minimum age considered an adult in their country (either aged 16 years and older or aged 18 years and older), and had significant (defined as at risk of bleeding to death) upper or lower gastrointestinal bleeding. Patients were randomly assigned by selection of a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients received either a loading dose of 1 g tranexamic acid, which was added to 100 mL infusion bag of 0·9% sodium chloride and infused by slow intravenous injection over 10 min, followed by a maintenance dose of 3 g tranexamic acid added to 1 L of any isotonic intravenous solution and infused at 125 mg/h for 24 h, or placebo (sodium chloride 0·9%). Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was death due to bleeding within 5 days of randomisation; analysis excluded patients who received neither dose of the allocated treatment and those for whom outcome data on death were unavailable. This trial was registered with Current Controlled Trials, ISRCTN11225767, and ClinicalTrials.gov, NCT01658124.FindingsBetween July 4, 2013, and June 21, 2019, we randomly allocated 12 009 patients to receive tranexamic acid (5994, 49·9%) or matching placebo (6015, 50·1%), of whom 11 952 (99·5%) received the first dose of the allocated treatment. Death due to bleeding within 5 days of randomisation occurred in 222 (4%) of 5956 patients in the tranexamic acid group and in 226 (4%) of 5981 patients in the placebo group (risk ratio [RR] 0·99, 95% CI 0·82–1·18). Arterial thromboembolic events (myocardial infarction or stroke) were similar in the tranexamic acid group and placebo group (42 [0·7%] of 5952 vs 46 [0·8%] of 5977; 0·92; 0·60 to 1·39). Venous thromboembolic events (deep vein thrombosis or pulmonary embolism) were higher in tranexamic acid group than in the placebo group (48 [0·8%] of 5952 vs 26 [0·4%] of 5977; RR 1·85; 95% CI 1·15 to 2·98).InterpretationWe found that tranexamic acid did not reduce death from gastrointestinal bleeding. On the basis of our results, tranexamic acid should not be used for the treatment of gastrointestinal bleeding outside the context of a randomised trial.</div