Analysis of the Most Popular Online Ankle Fracture-Related Patient Education Materials.

Background: Given the increasing accessibility of Internet access, it is critical to ensure that the informational material available online for patient education is both accurate and readable to promote a greater degree of health literacy. This study sought to investigate the quality and readability of the most popular online resources for ankle fractures. Methods: After conducting a Google search using 6 terms related to ankle fractures, we collected the first 20 nonsponsored results for each term. Readability was evaluated using the Flesch Reading Ease (FRE), Flesch-Kincaid Grade Level (FKGL), and Gunning Fog Index (GFI) instruments. Quality was evaluated using custom created Ankle Fracture Index (AFI). Results: A total of 46 of 120 articles met the inclusion criteria. The mean FKGL, FRE, and GFI scores were 8.4 ± 0.5, 57.5 ± 3.2, and 10.5 ± 0.5, respectively. The average AFI score was 15.4 ± 1.4, corresponding to an "acceptable" quality rating. Almost 70% of articles (n = 32) were written at or below the recommended eighth-grade reading level. Most articles discussed the need for imaging in diagnosis and treatment planning while neglecting to discuss the risks of surgery or potential future operations. Conclusion: We found that online patient-facing materials on ankle fractures demonstrated an eighth-grade average reading grade level and an acceptable quality on content analysis. Further work should surround increasing information regarding risk factors, complications for surgery, and long-term recovery while ensuring that readability levels remain below at least the eighth-grade level.

A predictive model for ethylene-mediated auxin and cytokinin patterning in the Arabidopsis root.

The interaction between auxin and cytokinin is important in many aspects of plant development. Experimental measurements of both auxin and cytokinin concentration and reporter gene expression clearly show the coexistence of auxin and cytokinin concentration patterning in Arabidopsis root development. However, in the context of crosstalk among auxin, cytokinin, and ethylene, little is known about how auxin and cytokinin concentration patterns simultaneously emerge and how they regulate each other in the Arabidopsis root. This work utilizes a wide range of experimental observations to propose a mechanism for simultaneous patterning of auxin and cytokinin concentrations. In addition to revealing the regulatory relationships between auxin and cytokinin, this mechanism shows that ethylene signaling is an important factor in achieving simultaneous auxin and cytokinin patterning, while also predicting other experimental observations. Combining the mechanism with a realistic in silico root model reproduces experimental observations of both auxin and cytokinin patterning. Predictions made by the mechanism can be compared with a variety of experimental observations, including those obtained by our group and other independent experiments reported by other groups. Examples of these predictions include patterning of auxin biosynthesis rate, changes in PIN1 and PIN2 patterns in pin3,4,7 mutants, changes in cytokinin patterning in the pls mutant, PLS patterning, and various trends in different mutants. This research reveals a plausible mechanism for simultaneous patterning of auxin and cytokinin concentrations in Arabidopsis root development and suggests a key role for ethylene pattern integration.

Yeast lacking the sterol C-5 desaturase Erg3 are tolerant to the anti-inflammatory triterpenoid saponin escin.

Escin is a mixture of over 30 glycosylated triterpenoid (saponin) structures, extracted from the dried fruit of horse chestnuts. Escin is currently used as an anti-inflammatory, and has potential applications in the treatment of arthritis and cancer. Engineered yeast would enable production of specific bioactive components of escin at industrial scale, however many saponins have been shown to be toxic to yeast. Here we report that a Saccharomyces cerevisiae strain specifically lacking the sterol C-5 desaturase gene ERG3, exhibits striking enhanced tolerance to escin treatment. Transcriptome analyses, as well as pre-mixing of escin with sterols, support the hypothesis that escin interacts directly with ergosterol, but not as strongly with the altered sterols present in erg3Δ. A diverse range of saponins are of commercial interest, and this research highlights the value of screening lipidome mutants to identify appropriate hosts for engineering the industrial production of saponins.

Insights from linking police domestic abuse data and health data in South Wales, UK: a linked routine data analysis using decision tree classification.

BACKGROUND: Exposure to domestic abuse can lead to long-term negative impacts on the victim's physical and psychological wellbeing. The 1998 Crime and Disorder Act requires agencies to collaborate on crime reduction strategies, including data sharing. Although data sharing is feasible for individuals, rarely are whole-agency data linked. This study aimed to examine the knowledge obtained by integrating information from police and health-care datasets through data linkage and analyse associated risk factor clusters. METHODS: This retrospective cohort study analyses data from residents of South Wales who were victims of domestic abuse resulting in a Public Protection Notification (PPN) submission between Aug 12, 2015 and March 31, 2020. The study links these data with the victims' health records, collated within the Secure Anonymised Information Linkage databank, to examine factors associated with the outcome of an Emergency Department attendance, emergency hospital admission, or death within 12 months of the PPN submission. To assess the time to outcome for domestic abuse victims after the index PPN submission, we used Kaplan-Meier survival analysis. We used multivariable Cox regression models to identify which factors contributed the highest risk of experiencing an outcome after the index PPN submission. Finally, we created decision trees to describe specific groups of individuals who are at risk of experiencing a domestic abuse incident and subsequent outcome. FINDINGS: After excluding individuals with multiple PPN records, duplicates, and records with a poor matching score or missing fields, the resulting clean dataset consisted of 8709 domestic abuse victims, of whom 6257 (71·8%) were female. Within a year of a domestic abuse incident, 3650 (41·9%) individuals had an outcome. Factors associated with experiencing an outcome within 12 months of the PPN included younger victim age (hazard ratio 1·183 [95% CI 1·053-1·329], p=0·0048), further PPN submissions after the initial referral (1·383 [1·295-1·476]; p<0·0001), injury at the scene (1·484 [1·368-1·609]; p<0·0001), assessed high risk (1·600 [1·444-1·773]; p<0·0001), referral to other agencies (1·518 [1·358-1·697]; p<0·0001), history of violence (1·229 [1·134-1·333]; p<0·0001), attempted strangulation (1·311 [1·148-1·497]; p<0·0001), and pregnancy (1·372 [1·142-1·648]; p=0·0007). Health-care data before the index PPN established that previous Emergency Department and hospital admissions, smoking, smoking cessation advice, obstetric codes, and prescription of antidepressants and antibiotics were associated with having a future outcome following a domestic abuse incident. INTERPRETATION: The results indicate that vulnerable individuals are detectable in multiple datasets before and after involvement of the police. Operationalising these findings could reduce police callouts and future Emergency Department or hospital admissions, and improve outcomes for those who are vulnerable. Strategies include querying previous Emergency Department and hospital admissions, giving a high-risk assessment for a pregnant victim, and facilitating data linkage to identify vulnerable individuals. FUNDING: National Institute for Health Research.

Weighting of risk factors for low birth weight: a linked routine data cohort study in Wales, UK.

OBJECTIVE: Globally, 20 million children are born with a birth weight below 2500 g every year, which is considered as a low birthweight (LBW) baby. This study investigates the contribution of modifiable risk factors in a nationally representative Welsh e-cohort of children and their mothers to inform opportunities to reduce LBW prevalence. DESIGN: A longitudinal cohort study based on anonymously linked, routinely collected multiple administrative data sets. PARTICIPANTS: The cohort, (N=693 377) comprising of children born between 1 January 1998 and 31 December 2018 in Wales, was selected from the National Community Child Health Database. OUTCOME MEASURES: The risk factors associated with a binary LBW (outcome) variable were investigated with multivariable logistic regression (MLR) and decision tree (DT) models. RESULTS: The MLR model showed that non-singleton children had the highest risk of LBW (adjusted OR 21.74 (95% CI 21.09 to 22.40)), followed by pregnancy interval less than 1 year (2.92 (95% CI 2.70 to 3.15)), maternal physical and mental health conditions including diabetes (2.03 (1.81 to 2.28)), anaemia (1.26 (95% CI 1.16 to 1.36)), depression (1.58 (95% CI 1.43 to 1.75)), serious mental illness (1.46 (95% CI 1.04 to 2.05)), anxiety (1.22 (95% CI 1.08 to 1.38)) and use of antidepressant medication during pregnancy (1.92 (95% CI 1.20 to 3.07)). Additional maternal risk factors include smoking (1.80 (95% CI 1.76 to 1.84)), alcohol-related hospital admission (1.60 (95% CI 1.30 to 1.97)), substance misuse (1.35 (95% CI 1.29 to 1.41)) and evidence of domestic abuse (1.98 (95% CI 1.39 to 2.81)). Living in less deprived area has lower risk of LBW (0.70 (95% CI 0.67 to 0.72)). The most important risk factors from the DT models include maternal factors such as smoking, maternal weight, substance misuse record, maternal age along with deprivation-Welsh Index of Multiple Deprivation score, pregnancy interval and birth order of the child. CONCLUSION: Resources to reduce the prevalence of LBW should focus on improving maternal health, reducing preterm births, increasing awareness of what is a sufficient pregnancy interval, and to provide adequate support for mothers' mental health and well-being.

An overview of the state of the regulatory and preclinical requirements for nanomaterials including medical devices.

Nanomaterials when exposed to biological systems may produce oxidative stress, genotoxicity, and carcinogenicity, as nanoparticle toxicity is speculated to be different from larger particles of the same chemical composition due to the change of their physical properties from their smaller size and resultant higher surface area. Regulatory frameworks have become established to address the influx of nanomaterials to assess the potential benefit to human health but also where there is a risk to both human health and the environment. This article focuses on these regulatory frameworks, including the risk from inhalation, where these changes have already been implemented and reflected in the revised Organisation for Economic Co-operation and Development OECD 412 and OECD 413 guidelines as additional investigations and endpoints have been added to determine the risk posed by this novel group of materials. Integr Environ Assess Manag 2021;17:1098-1104. © 2021 SETAC.

The Enduring Effects of Parental Alcohol, Tobacco, and Drug Use on Child Well-being: A Multilevel Meta-Analysis.

The effects of psychoactive substance abuse are not limited to the user, but extend to the entire family system, with children of substance abusers being particularly at risk. This meta-analysis attempted to quantify the longitudinal relationship between parental alcohol, tobacco, and drug use and child well-being, investigating variation across a range of substance and well-being indices and other potential moderators. We performed a literature search of peer-reviewed, English language, longitudinal observational studies that reported outcomes for children aged 0 to 18 years. In total, 56 studies, yielding 220 dependent effect sizes, met inclusion criteria. A multilevel random-effects model revealed a statistically significant, small detriment to child well-being for parental substance abuse over time (r = .15). Moderator analyses demonstrated that the effect was more pronounced for parental drug use (r = .25), compared with alcohol use (r = .13), tobacco use (r = .13), and alcohol use disorder (r = .14). Results highlight a need for future studies that better capture the effect of parental psychoactive substance abuse on the full breadth of childhood well-being outcomes and to integrate substance abuse into models that specify the precise conditions under which parental behavior determines child well-being.Registration: PROSPERO CRD42017076088.

Drinking beer, wine or spirits - does it matter for inequalities in alcohol-related hospital admission? A record-linked longitudinal study in Wales.

BACKGROUND: Alcohol-related harm has been found to be higher in disadvantaged groups, despite similar alcohol consumption to advantaged groups. This is known as the alcohol harm paradox. Beverage type is reportedly socioeconomically patterned but has not been included in longitudinal studies investigating record-linked alcohol consumption and harm. We aimed to investigate whether and to what extent consumption by beverage type, BMI, smoking and other factors explain inequalities in alcohol-related harm. METHODS: 11,038 respondents to the Welsh Health Survey answered questions on their health and lifestyle. Responses were record-linked to wholly attributable alcohol-related hospital admissions (ARHA) eight years before the survey month and until the end of 2016 within the Secure Anonymised Information Linkage (SAIL) Databank. We used survival analysis, specifically multi-level and multi-failure Cox mixed effects models, to calculate the hazard ratios of ARHA. In adjusted models we included the number of units consumed by beverage type and other factors, censoring for death or moving out of Wales. RESULTS: People living in more deprived areas had a higher risk of admission (HR 1.75; 95% CI 1.23-2.48) compared to less deprived. Adjustment for the number of units by type of alcohol consumed only reduced the risk of ARHA for more deprived areas by 4% (HR 1.72; 95% CI 1.21-2.44), whilst adding smoking and BMI reduced these inequalities by 35.7% (HR 1.48; 95% CI 1.01-2.17). These social patterns were similar for individual-level social class, employment, housing tenure and highest qualification. Inequalities were further reduced by including either health status (16.6%) or mental health condition (5%). Unit increases of spirits drunk were positively associated with increasing risk of ARHA (HR 1.06; 95% CI 1.01-1.12), higher than for other drink types. CONCLUSIONS: Although consumption by beverage type was socioeconomically patterned, it did not help explain inequalities in alcohol-related harm. Smoking and BMI explained around a third of inequalities, but lower socioeconomic groups had a persistently higher risk of (multiple) ARHA. Comorbidities also explained a further proportion of inequalities and need further investigation, including the contribution of specific conditions. The increased harms from consumption of stronger alcoholic beverages may inform public health policy.

Method and Apparatus for the Automated Delivery of Continuous Neural Stem Cell Trails Into the Spinal Cord of Small and Large Animals.

BACKGROUND: Immature neurons can extend processes after transplantation in adult animals. Neuronal relays can form between injected neural stem cells (NSCs) and surviving neurons, possibly improving recovery after spinal cord injury (SCI). Cell delivery methods of single or multiple bolus injections of concentrated cell suspensions thus far tested in preclinical and clinical experiments are suboptimal for new tract formation. Nonuniform injectate dispersal is often seen due to gravitational cell settling and clumping. Multiple injections have additive risks of hemorrhage, parenchymal damage, and cellular reflux and require additional surgical exposure. The deposition of multiply delivered cells boluses may be uneven and discontinuous. OBJECTIVE: To develop an injection apparatus and methodology to deliver continuous cellular trails bridging spinal cord lesions. METHODS: We improved the uniformity of cellular trails by formulating NSCs in hyaluronic acid. The TrailmakerTM stereotaxic injection device was automatized to extend a shape memory needle from a single-entry point in the spinal cord longitudinal axis to "pioneer" a new trail space and then retract while depositing an hyaluronic acid-NSC suspension. We conducted testing in a collagen spinal models, and animal testing using human NSCs (hNSCs) in rats and minipigs. RESULTS: Continuous surviving trails of hNSCs within rat and minipig naive spinal cords were 12 and 40 mm in length. hNSC trails were delivered across semi-acute contusion injuries in rats. Transplanted hNSCs survived and were able to differentiate into neural lineage cells and astrocytes. CONCLUSION: The TrailmakerTM creates longitudinal cellular trails spanning multiple levels from a single-entry point. This may enhance the ability of therapeutics to promote functional relays after SCI.

The relationship between alcohol use and long-term cognitive decline in middle and late life: a longitudinal analysis using UK Biobank.

Background: Using UK Biobank data, this study sought to explain the causal relationship between alcohol intake and cognitive decline in middle and older aged populations. Methods: Data from 13 342 men and women, aged between 40 and 73 years were used in regression analysis that tested the functional relationship and impact of alcohol on cognitive performance. Performance was measured using mean reaction time (RT) and intra-individual variation (IIV) in RT, collected in response to a perceptual matching task. Covariates included body mass index, physical activity, tobacco use, socioeconomic status, education and baseline cognitive function. Results: A restricted cubic spline regression with three knots showed how the linear (ß1 = -0.048, 95% CI: -0.105 to -0.030) and non-linear effects (ß2 = 0.035, 95% CI: 0.007-0.059) of alcohol use on mean RT and IIV in RT (ß1 = -0.055, 95% CI: -0.125 to -0.034; ß2 = 0.034, 95% CI: 0.002-0.064) were significant adjusting for covariates. Cognitive function declined as alcohol use increased beyond 10 g/day. Decline was more apparent as age increased. Conclusions: The relationship between alcohol use and cognitive function is non-linear. Consuming more than one UK standard unit of alcohol per day is detrimental to cognitive performance and is more pronounced in older populations.

Feasibility of alcohol misuse screening and treatment in the dental setting.

BACKGROUND: Dental specialists treat conditions such as facial trauma and oral cancer that can result from alcohol misuse. Visits to primary dental care professionals are oriented towards prevention. Interventions coordinated by specialist services but delivered strategically in primary care could therefore potentially help to reduce burdens on secondary care services. The aim of this study was to determine the feasibility of screening for alcohol misuse and providing brief intervention in a primary dental care setting. METHODS: In this randomised controlled trial, patients aged 18-65 years were recruited from a local general dental practice. Patients were stratified according to appointment (with a dentist or hygienist). Reception staff administered envelope packs containing screening materials (the Modified Single Alcohol Screening Question [M-SASQ]), consent forms, and a short survey collecting contact details to patients who agreed to take part in the study. Packs were randomly pre-allocated to control and intervention groups by strata using block randomisation before the start of the study. Consenting patients scoring positively on the M-SASQ for drinking hazardously and allocated to the intervention group received a motivational intervention to reduce alcohol intake from either the hygienist or dentist. Patients in the control group received usual care. The outcome assessor and patients were masked to allocations. The outcome measure at 3 months was M-SASQ score. This trial is registered with the ISRCTN registry, number ISRCTN18745862. FINDINGS: One hygiene patient and 106 dental patients were recruited. The hygiene patient did not score positively on the M-SASQ for alcohol misuse. Of the 106 dental patients, 46 (43%) scored positively, with 26 allocated to the intervention group and 20 to the control group. Follow-up data were available for 22 (48%) of the 46 patients (12 intervention, 10 control). M-SASQ scores changed from positive to negative for two patients in the intervention and five in the control group. INTERPRETATION: Alcohol misuse screening and treatment was feasible in a primary dental care setting; this suggests a new approach involving the general dental team, which could potentially reduce burdens on specialist dental services. Overall, in this practice, the dentist was best placed to deliver the intervention rather than the hygienist since these health-care professionals saw most of the patients recruited into the trial. Contamination might have been a problem because more patients in the control group changed M-SASQ score. Building on these findings, a multicentre, cluster randomised controlled trial is planned. FUNDING: Royal College of Surgeons of England.

Some fundamental aspects of modeling auxin patterning in the context of auxin-ethylene-cytokinin crosstalk.

The activities of hormones in the Arabidopsis root depend on cellular context and exhibit either synergistic or antagonistic interactions. Patterning in Arabidopsis root development is coordinated via a localized auxin concentration maximum in the root tip, mediating transcription of key regulatory genes. Auxin concentration and response are each regulated by diverse interacting hormones and gene expression and therefore cannot change independently of those hormones and genes. For example, experimental data accumulated over many years have shown that both ethylene and cytokinin regulate auxin concentration and response. Using the crosstalk of auxin-ethylene-cytokinin as a paradigm, we discuss the links between experimental data, reaction kinetics and spatiotemporal modeling to dissect hormonal crosstalk. In particular, we discuss how kinetic equations for modeling auxin concentration are formulated based on experimental data and also the underlying assumptions for deriving those kinetic equations. Furthermore, we show that, by integrating kinetic equations with spatial root structure, modeling of spatiotemporal hormonal crosstalk is a powerful tool for analyzing and predicting the roles of multiple hormone interactions in auxin patterning. Finally, we summarize important considerations in developing a spatiotemporal hormonal crosstalk model for plant root development.

Children's toothbrushing frequency: the influence of parents' rationale for brushing, habits and family routines.

OBJECTIVES: To assess the relationship between parental and family factors and children's toothbrushing frequency at different times of day. METHODS: A cross-sectional questionnaire survey of predominantly low-socio-economic status parents of children aged 3-6 years (n = 296) in South Wales, UK. Data were collected on the child's weekly toothbrushing frequency (morning and evening), the parents' rationale for brushing their child's teeth in the morning and evening, the strength of a parent's habit for brushing a child's teeth in the morning and evening, and the extent to which the family's daily routines were stable from day to day. Socio-demographic details were also collected. RESULTS: Reported weekly brushing frequency was significantly (p < 0.001) higher in the morning (mean ± SD: 6.57 ± 1.37) than the evening (mean ± SD: 5.99 ± 2.15). Parents had significantly (p < 0.001) more interest in the cosmetic benefits of toothbrushing in the morning compared to the evening. Multivariate analysis showed that an increasing focus on the cosmetic benefits of toothbrushing was associated with significantly (p < 0.05) less weekly brushing in the evening. The extent to which brushing a child's teeth was 'habitual' was significantly (p < 0.001) associated with weekly toothbrushing frequency at both times of day. CONCLUSIONS: Parents' rationale for brushing their children's teeth can vary at both an individual level and at different times of day. Understanding these variations is important in designing interventions to improve brushing frequency for at-risk children. The results also demonstrate habituation as being an important factor in understanding toothbrushing frequency. Further research is required to understand the mechanisms involved in habit formation and maintenance with children's oral hygiene behaviour.

Towards a cell factory for vitamin B12 production in Bacillus megaterium: bypassing of the cobalamin riboswitch control elements.

Bacillus megaterium is a bacterium that has been used in the past for the industrial production of vitamin B12 (cobalamin), the anti-pernicious anaemia factor. Cobalamin is a modified tetrapyrrole with a cobalt ion coordinated within its macrocycle. More recently, B. megaterium has been developed as a host for the high-yield production of recombinant proteins using a xylose inducible promoter system. Herein, we revisit cobalamin production in B. megaterium DSM319. We have investigated the importance of cobalt for optimum growth and cobalamin production. The cobaltochelatase (CbiX(L)) is encoded within a 14-gene cobalamin biosynthetic (cbi) operon, whose gene-products oversee the transformation of uroporphyrinogen III into adenosylcobyrinic acid a,c-diamide, a key precursor of cobalamin synthesis. The production of CbiX(L) in response to exogenous cobalt was monitored. The metal was found to stimulate cobalamin biosynthesis and decrease the levels of CbiX(L). From this we were able to show that the entire cbi operon is transcriptionally regulated by a B12-riboswitch, with a switch-off point at approximately 5 nM cobalamin. To bypass the effects of the B12-riboswitch the cbi operon was cloned without these regulatory elements. Growth of these strains on minimal media supplemented with glycerol as a carbon source resulted in significant increases in cobalamin production (up to 200 µg L(-1)). In addition, a range of partially amidated intermediates up to adenosylcobyric acid was detected. These findings outline a potential way to develop B. megaterium as a cell factory for cobalamin production using cheap raw materials.

N-WASP-directed actin polymerization activates Cas phosphorylation and lamellipodium spreading.

Tyrosine phosphorylation of the substrate domain of Cas (CasSD) correlates with increased cell migration in healthy and diseased cells. Here, we address the mechanism leading to the phosphorylation of CasSD in the context of fibronectin-induced early spreading of fibroblasts. We have previously demonstrated that mechanical stretching of CasSD exposes phosphorylation sites for Src family kinases (SFKs). Surprisingly, phosphorylation of CasSD was independent of myosin contractile activity but dependent on actin polymerization. Furthermore, we found that CasSD phosphorylation in the early stages of cell spreading required: (1) integrin anchorage and integrin-mediated activation of SFKs, (2) association of Cas with focal adhesion kinase (FAK), and (3) N-WASP-driven actin-assembly activity. These findings, and analyses of the interactions of the Cas domains, indicate that the N-terminus of Cas associates with the FAK-N-WASP complex at the protrusive edge of the cell and that the C-terminus of Cas associates with the immobilized integrin-SFK cluster. Thus, extension of the leading edge mediated by actin polymerization could stretch Cas during early cell spreading, priming it for phosphorylation.

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).

It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification.

Netrin-1 attracts axons through FAK-dependent mechanotransduction.

The mechanism by which extracellular cues influence intracellular biochemical cascades that guide axons is important, yet poorly understood. Because of the mechanical nature of axon extension, we explored whether the physical interactions of growth cones with their guidance cues might be involved. In the context of mouse spinal commissural neuron axon attraction to netrin-1, we found that mechanical attachment of netrin-1 to the substrate was required for axon outgrowth, growth cone expansion, axon attraction and phosphorylation of focal adhesion kinase (FAK) and Crk-associated substrate (CAS). Myosin II activity was necessary for traction forces >30 pN on netrin-1. Interestingly, while these myosin II-dependent forces on netrin-1 substrates or beads were needed to increase the kinase activity and phosphorylation of FAK, they were not necessary for netrin-1 to increase CAS phosphorylation. When FAK kinase activity was inhibited, the growth cone's ability to recruit additional adhesions and to generate forces >60 pN on netrin-1 was disrupted. Together, these findings demonstrate an important role for mechanotransduction during chemoattraction to netrin-1 and that mechanical activation of FAK reinforces interactions with netrin-1 allowing greater forces to be exerted.

Traction on immobilized netrin-1 is sufficient to reorient axons.

During embryonic development, axons are guided to their target by patterning proteins encountered along their trajectory. These cues can be linked to the cells that produce them or secreted into the extracellular matrix. Whether secreted cues, like netrin-1, provide traction for the growth cone when they become attached to the extracellular matrix is unclear. Advancing spinal commissural neuron growth cones were shown to generate local forces of 4 to 15 piconewtons but, when confronted with immobilized netrin-1, generated traction forces in excess of 63 piconewtons on netrin-1 that can redirect the trajectory of the axon.

Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1.

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear. Here, we show that netrin 1, through its receptor DCC, inhibits RhoA in embryonic spinal commissural neurons. To determine whether netrin 1-mediated chemoattraction requires Rho function, we inhibited Rho signaling and assayed axon outgrowth and turning towards netrin 1. Additionally, we examined two important mechanisms that influence the guidance of axons to netrin 1: substrate adhesion and transport of the netrin receptor DCC to the plasma membrane. We found that inhibiting Rho signaling increased plasma membrane DCC and adhesion to substrate-bound netrin 1, and also enhanced netrin 1-mediated axon outgrowth and chemoattractive axon turning. Conversely, overexpression of RhoA or constitutively active RhoA inhibited axonal responses to netrin 1. These findings provide evidence that Rho signaling reduces axonal chemoattraction to netrin 1 by limiting the amount of plasma membrane DCC at the growth cone, and suggest that netrin 1-mediated inhibition of RhoA activates a positive-feedback mechanism that facilitates chemoattraction to netrin 1. Notably, these findings also have relevance for CNS regeneration research. Inhibiting RhoA promotes axon regeneration by disrupting inhibitory responses to myelin and the glial scar. By contrast, we demonstrate that axon chemoattraction to netrin 1 is not only maintained but enhanced, suggesting that this might facilitate directing regenerating axons to appropriate targets.

Soluble adenylyl cyclase is not required for axon guidance to netrin-1.

During development, axons are directed to their targets by extracellular guidance cues. The axonal response to the guidance cue netrin-1 is profoundly influenced by the concentration of cAMP within the growth cone. In some cases, cAMP affects the sensitivity of the growth cone to netrin-1, whereas in others it changes the response to netrin-1 from attraction to repulsion. The effects of cAMP on netrin-1 action are well accepted, but the critical issue of whether cAMP production is activated by a netrin-1 induced signaling cascade remains uncertain. A previous report has suggested that axon guidance in response to netrin-1 requires cAMP production mediated by soluble adenyl cyclase (sAC). We have used genetic, molecular and biochemical strategies to assess this issue. Surprisingly, we found only extremely weak expression of sAC in embryonic neurons and determined that, under conditions where netrin-1 directs axonal pathfinding, exposure to netrin-1 does not alter cAMP levels. Furthermore, although netrin-1-deficient mice exhibit major axon guidance defects, we show that pathfinding is normal in sAC-null mice. Therefore, although cAMP can alter the response of axons to netrin-1, we conclude that netrin-1 does not alter cAMP levels in axons attracted by this cue, and that sAC is not required for axon attraction to netrin-1.