The effect of increased fruit and vegetable consumption on blood pressure and lipids: a pooled analysis of six randomised controlled fruit and vegetable intervention trials.

BACKGROUND: Increasing fruit and vegetable (FV) consumption is associated with reduced cardiovascular disease risk in observational studies but with little evidence from randomised controlled trials (RCTs). The impact of concurrent pharmacological therapy is unknown. OBJECTIVE: To pool data from six RCTs to examine the effect of increasing FV intake on blood pressure (BP) and lipid profile, also exploring whether effects differed by medication use. DESIGN: Across trials, dietary intake was assessed by diet diaries or histories, lipids by routine biochemical methods and BP by automated monitors. Linear regression provided an estimate of the change in lipid profile or BP associated with a one portion increase in self-reported daily FV intake, with interaction terms fitted for medication use. RESULTS: The pooled sample included a total of 554 participants (308 males and 246 females). Meta-analysis of regression coefficients revealed no significant change in either systolic or diastolic BP per portion FV increase, although there was significant heterogeneity across trials for systolic BP (I2 = 73%). Neither adjusting for change in body mass index, nor analysis according to use of anti-hypertensive medication altered the relationship. There was no significant change in lipid profile per portion FV increase, although there was a significant reduction in total cholesterol among those not on lipid-lowering therapy (P < 0.05 after Bonferroni correction). CONCLUSION: Pooled analysis of six individual FV trials showed no impact of increasing intake on BP or lipids, but there was a total cholesterol-lowering effect in those not on lipid-lowering therapy.

POLARISED views and FRETting about probe modulation assays: Learning from High Throughput Screening.

Fluorescent probe modulation assays are a widely used approach to monitor displacement or stabilisation of fluorescently labelled tool ligands by test compounds. These assays allow an optical read-out of probe-receptor binding and can be used to detect compounds that compete with the labelled ligand, either directly or indirectly. Probes for both orthosteric and allosteric sites are often employed. The method can also be used to identify test compounds that may stabilise the ternary complex, offering an opportunity to discover novel molecular glues. The utility of these fluorescence-based assays within high-throughput screening has been facilitated by the use of streptavidin labelled terbium as a donor and access to a range of different acceptor fluorophores. During 2023, the High-throughput Screening group at AstraZeneca carried out 8 high-throughput screens using these approaches. In this manuscript we will present the types of assays used, an overview of the timelines for assay development and screening, the application of orthogonal artefact methods to aid hit finding and the results of the screens in terms of hit rate and the number of compounds identified with IC50 values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented.

Bacterial Emission Factors: A Foundation for the Terrestrial-Atmospheric Modeling of Bacteria Aerosolized by Wildland Fires.

Wildland fire is a major global driver in the exchange of aerosols between terrestrial environments and the atmosphere. This exchange is commonly quantified using emission factors or the mass of a pollutant emitted per mass of fuel burned. However, emission factors for microbes aerosolized by fire have yet to be determined. Using bacterial cell concentrations collected on unmanned aircraft systems over forest fires in Utah, USA, we determine bacterial emission factors (BEFs) for the first time. We estimate that 1.39 × 1010 and 7.68 × 1011 microbes are emitted for each Mg of biomass consumed in fires burning thinning residues and intact forests, respectively. These emissions exceed estimates of background bacterial emissions in other studies by 3-4 orders of magnitude. For the ∼2631 ha of similar forests in the Fishlake National Forest that burn each year on average, an estimated 1.35 × 1017 cells or 8.1 kg of bacterial biomass were emitted. BEFs were then used to parametrize a computationally scalable particle transport model that predicted over 99% of the emitted cells were transported beyond the 17.25 x 17.25 km model domain. BEFs can be used to expand understanding of global wildfire microbial emissions and their potential consequences to ecosystems, the atmosphere, and humans.

Discovery, Optimization, and Biological Evaluation of Arylpyridones as Cbl-b Inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.

Discovery of a Novel Benzodiazepine Series of Cbl-b Inhibitors for the Enhancement of Antitumor Immunity.

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ligase that is responsible for repressing T-cell, natural killer (NK) cell, and B-cell activation. The robust antitumor activity observed in Cbl-b deficient mice arising from elevated T-cell and NK-cell activity justified our discovery effort toward Cbl-b inhibitors that might show therapeutic promise in immuno-oncology, where activation of the immune system can drive the recognition and killing of cancer cells. We undertook a high-throughput screening campaign followed by structure-enabled optimization to develop a novel benzodiazepine series of potent Cbl-b inhibitors. This series displayed nanomolar levels of biochemical potency, as well as potent T-cell activation. The functional activity of this class of Cbl-b inhibitors was further corroborated with ubiquitin-based cellular assays.

A comparative study of outcomes of burns across multiple levels of care.

BACKGROUND: Burn injuries are a significant contributor to the burden of diseases. The management of burns at specialised burn centres has been shown to improve survival. However, in low- and middle-income countries (LMICs) major burns are managed at non-specialised burn centres due to resource constraints. There is insufficient data on survival from treatment at non-specialised burn centres in LMICs. This study aimed to compare the outcomes of burns treatment between a specialised burn centre and five non-specialised centres. METHODS: A prospective cohort study was conducted on patients aged 18 years or above from January 1, 2021 to September 30, 2021. Participants were selected from the admission register at the emergency department. All burns irrespective of the mechanism of injury or %TBSA were included. Data were entered into REDCap. Statistical analysis of outcomes such as positive blood culture, length of hospital stay (LOHS) and 90-day mortality between specialised burn versus non-specialised centres was performed. Furthermore, an analysis of risk factors for mortality was performed and survival data computed. RESULTS: Of the 488 study participants, 36% were admitted to a specialised burn centre compared to 64% admitted to non-specialised centres. The demographic characteristics were similar between centres. Patients at the specialised burn centre compared to non-specialised centres had a significantly higher inhalation injury of 30.9% vs 7.7% (p < 0.001), > 10%TBSA at 83.4% vs 45.7% (p < 0.001), > 20%TBSA at 46.9% vs 16.6% (p < 0.001), and a median (IQR) ABSI score of 6 (5-7) vs 5 (4-6) (p < 0.0001). Furthermore, patients from specialised burn vs non-specialised centres had a longer median (IQR) time from injury to first burn excision at 7 (4-11) vs 5 (2-10) days, higher rate of burn sepsis 69% vs 35%, increased LOHS 17 (11-27) vs 12 (6-22) days, and 90-day mortality rates at 19.4% vs 6.4%. After adjusting for cofounding variables, survival data showed no difference between specialised burn and non-specialised centres (HR 1.8 95% CI 1.0-3.2, p = 0.05). CONCLUSION: Although it appears that the survival of burn patients managed at non-specialised centres in a middle-income country is comparable to those managed at specialised burn centres, there is uncounted bias in our survival data. Hence, a change in practice is not advocated. However, due to resource constraint specialised burn centres in addition to managing major burns should provide training and support to the non-specialised centres.

Unveiling metabolic pathways involved in the extreme desiccation tolerance of an Atacama cyanobacterium.

Gloeocapsopsis dulcis strain AAB1 is an extremely xerotolerant cyanobacterium isolated from the Atacama Desert (i.e., the driest and oldest desert on Earth) that holds astrobiological significance due to its ability to biosynthesize compatible solutes at ultra-low water activities. We sequenced and assembled the G. dulcis genome de novo using a combination of long- and short-read sequencing, which resulted in high-quality consensus sequences of the chromosome and two plasmids. We leveraged the G. dulcis genome to generate a genome-scale metabolic model (iGd895) to simulate growth in silico. iGd895 represents, to our knowledge, the first genome-scale metabolic reconstruction developed for an extremely xerotolerant cyanobacterium. The model's predictive capability was assessed by comparing the in silico growth rate with in vitro growth rates of G. dulcis, in addition to the synthesis of trehalose. iGd895 allowed us to explore simulations of key metabolic processes such as essential pathways for water-stress tolerance, and significant alterations to reaction flux distribution and metabolic network reorganization resulting from water limitation. Our study provides insights into the potential metabolic strategies employed by G. dulcis, emphasizing the crucial roles of compatible solutes, metabolic water, energy conservation, and the precise regulation of reaction rates in their adaptation to water stress.

Fractionated vs single-dose gemtuzumab ozogamicin with determinants of benefit in older patients with AML: the UK NCRI AML18 trial.

Addition of gemtuzumab ozogamicin (GO) to induction chemotherapy improves outcomes in older patients with acute myeloid leukemia (AML), but it is uncertain whether a fractionated schedule provides additional benefit to a single dose. We randomized 852 older adults (median age, 68-years) with AML/high-risk myelodysplasia to GO on day 1 (GO1) or on days 1 and 4 (GO2) of course 1 induction. The median follow-up period was 50.2 months. Although complete remission (CR) rates after course 1 did not significantly differ between arms (GO2, 63%; GO1, 57%; odds ratio [OR], 0.78; P = .08), there were significantly more patients who achieved CR with a measurable residual disease (MRD)<0.1% (50% vs 41%; OR, 0.72; P = .027). This differential MRD reduction with GO2 varied across molecular subtypes, being greatest for IDH mutations. The 5-year overall survival (OS) was 29% for patients in the GO2 arm and 24% for those in the GO1 arm (hazard ratio [HR], 0.89; P = .14). In a sensitivity analysis excluding patients found to have adverse cytogenetics or TP53 mutations, the 5-year OS was 33% for GO2 and 26% for GO1 (HR, 0.83; P = .045). In total, 228 (27%) patients received an allogeneic transplantation in first remission. Posttransplant OS was superior in the GO2 arm (HR, 0.67; P = .033); furthermore, the survival advantage from GO2 in the sensitivity analysis was lost when data of patients were censored at transplantation. In conclusion, GO2 was associated with a greater reduction in MRD and improved survival in older adults with nonadverse risk genetics. This benefit from GO2 was dependent on allogeneic transplantation to translate the better leukemia clearance into improved survival. This trial was registered at www.isrctn.com as #ISRCTN 31682779.

Barriers and facilitators of family rules and routines during pediatric cancer treatment.

OBJECTIVE: Pediatric nurses work closely with families of children with new cancer diagnoses and can provide essential supports to promote coping and adjustment. This cross-sectional qualitative study aimed to gather caregiver perspectives on barriers and facilitators to adaptive family functioning during the early phases of cancer treatment, with a focus on family rules and routines. METHODS: Caregivers (N = 44) of a child diagnosed with cancer and receiving active treatment completed a semi-structured interview about their engagement in family rules and routines. Time since diagnosis was abstracted from the medical record. A multi-pass inductive coding strategy was utilized to extract themes identifying caregiver-reported facilitators and barriers to maintaining consistent family rules and routines during the first year of pediatric treatment. RESULTS: Caregivers identified three primary contexts that presented barriers and facilitators to engagement in family rules and routines: the hospital setting (n = 40), the family system (n = 36), and the broader social and community setting (n = 26). Caregivers reported barriers primarily related to the demands of their child's treatment, additional caregiving needs, and needing to prioritize basic daily tasks (e.g., food, rest, household needs). Caregivers reported that different networks of support across contexts facilitated family rules and routines by expanding caregiver capacity in distinctive ways. CONCLUSIONS: Findings provided insight into the importance of having multiple networks of support to extend caregiving capacity in the context of cancer treatment demands. PRACTICE IMPLICATIONS: Providing nurses with training to facilitate problem-solving skills in the context of competing demands may provide a new avenue of clinical intervention at the bedside.

Scoping Review of Early Toxic Stress and Epigenetic Alterations in the Neonatal Intensive Care Unit.

BACKGROUND: Preterm infants are uniquely vulnerable to early toxic stress exposure while in the neonatal intensive care unit (NICU) and also being at risk for suboptimal neurodevelopmental outcomes. However, the complex biological mechanisms responsible for variations in preterm infants' neurodevelopmental outcomes because of early toxic stress exposure in the NICU remain unknown. Innovative preterm behavioral epigenetics research offers a possible mechanism and describes how early toxic stress exposure may lead to epigenetic alterations, potentially affecting short- and long-term outcomes. OBJECTIVE: The aim of this study was to review the relationships between early toxic stress exposures in the NICU and epigenetic alterations in preterm infants. The measurement of early toxic stress exposure in the NICU and effect of epigenetic alterations on neurodevelopmental outcomes in preterm infants were also examined. METHODS: We conducted a scoping review of the literature published between January 2011 and December 2021 using databases PubMed, CINAHL, Cochrance Library, PsycINFO, and Web of Science. Primary data-based research that examined epigenetics, stress, and preterm infants or NICU were included. RESULTS: A total of 13 articles from nine studies were included. DNA methylations of six specific genes were studied in relation to early toxic stress exposure in the NICU: SLC6A4, SLC6A3, OPRMI, NR3C1, HSD11B2, and PLAGL1. These genes are responsible for regulating serotonin, dopamine, and cortisol. Poorer neurodevelopmental outcomes were associated with alterations in DNA methylation of SLC6A4, NR3C1, and HSD11B2. Measurements of early toxic stress exposure in the NICU were inconsistent among the studies. DISCUSSION: Epigenetic alterations secondary to early toxic stress exposures in the NICU may be associated with future neurodevelopmental outcomes in preterm infants. Common data elements of toxic stress exposure in preterm infants are needed. Identification of the epigenome and mechanisms by which early toxic stress exposure leads to epigenetic alterations in this vulnerable population will provide evidence to design and test individualized intervention.

Risk and Resilient Functioning of Families of Children with Cancer during the COVID-19 Pandemic.

Previous literature highlights the impact of COVID-19 on family functioning. Less is known about the impact of the pandemic on families of pediatric cancer patients. In order to determine universal and unique risk and resilience factors of these families during the pandemic, a qualitative analysis was conducted on families currently receiving cancer treatment at a Midwestern hospital. Results of the data analysis depict ways in which these families have been impacted by and have adapted to COVID-19. These findings suggest that families of pediatric cancer patients have unique experiences in the context of COVID-19, in addition to universal experiences outlined in previous literature.

One Anastomosis Gastric Bypass Versus Single Anastomosis Duodenoileostomy with Sleeve: Comparative Analysis of 30-Day Outcomes Using the MBSAQIP.

INTRODUCTION/PURPOSE: One anastomosis gastric bypass (OAGB) and single anastomosis duodenoileostomy with sleeve (SADI-S) are two highly effective bariatric procedures that have been recently endorsed by the American Society of Metabolic and Bariatric Surgery (ASMBS). We compared the outcomes and safety profiles of SADI-S and OAGB using the Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (MBSAQIP) database. MATERIALS AND METHODS: Retrospective analysis on patients who underwent SADI-S or OAGB obtained from the MBSAQIP database 2020-2021. Patients who underwent concurrent procedures (besides EGD) or had missing data were removed. Variables included age, sex, body mass index, American Society of Anesthesiologists (ASA) class, and pertinent medical comorbidities. Data were analyzed for 30-day postoperative morbidity, mortality, reoperation, reintervention, and readmissions. p values were calculated using Student's t-test or Fisher analysis. RESULTS: A total of 694 and 1068 patients respectively underwent SADI-S or OAGB. Statistically significant comorbidities included age (42.2 ± 10.8 vs. 43.7 ± 12.2), BMI (50.6 ± 9.1 vs. 45.3 ± 7.1), ASA 2 (66 (9.5%) vs. 165 (15.4%)), ASA 4 [69 (9.9%) vs. 20 (1.9%)], and immunosuppressive therapy [24 (3.5%) vs. 17 (1.6%)]. Clavien-Dindo-based analysis highlighted that SADI-S had higher grade 2 (p = 0.005) and grade 4b (p = 0.001) complications. Patients who underwent SADI-S were twice as likely to be readmitted within 30 days (3.7% vs. 1.9%; p = 0.021). CONCLUSION: SADI-S had higher readmission rates and higher Clavien-Dindo grade 2 and 4b complications. To note, SADI-S patients had higher BMIs. Further studies are needed to determine the long-term complications and efficacy of both operations.

Predictors of mood or anxiety problems among transgender individuals seeking hormone therapy.

BACKGROUND: Mental health needs of transgender individuals can be complex with individual, social, and medical factors impacting symptoms. This study examines predictors of mood or anxiety problems among transgender individuals seeking hormone therapy (HT). METHODS: A retrospective chart review was conducted at 2 clinics providing gender-affirming HT. Cross-sectional data from initial patient encounters (N = 311) were used in this study. Bivariate correlations and multiple logistic regression analyses were carried out. RESULTS: Transgender women (TW) were 2.2 times more likely to have mood or anxiety problems while transgender men (TM) were 2.6 times more likely as the number of medical comorbidities increased. For both TW and TM, White race significantly increased the likelihood of mood or anxiety problems. Neither previous nor current HT were associated with mood or anxiety problems for TW and TM. However, receiving multiple gender-affirming procedures decreased the likelihood of mood or anxiety problems for TM. CONCLUSIONS: Gender-affirming care and addressing comorbidities can be important aspects of mental health needs for transgender individuals.

The majority of transgender men and women reported 1 or more chronic health conditions. These health conditions were associated with transgender individuals being more likely to have a mood or anxiety problem. Currently receiving or previously receiving hormonal therapy was not associated with mood or anxiety problems for transgender men or women, but having received 1 or multiple gender-affirming procedures was associated with a decrease in likelihood of having a mood or anxiety problem for transgender men. White race also was associated with increased likelihood of having a mood or anxiety problem for transgender men and women. These results highlight the need for primary care physicians to take a comprehensive approach when dealing with the mental health needs of transgender patients by ensuring that general health care needs are met while receiving gender-affirming care.

'They should walk with you': the perspectives of African Americans living with hypertension and their family members on disease self-management.

OBJECTIVES: African Americans are at significantly greater risk for hypertension, as well as worse hypertension-related morbidity and mortality than other racial/ethnic groups. Prior research aiming to address these health disparities has focused on improving individual patient self-management, with few studies testing family-centered interventions. We aimed to explore the perspectives of African Americans with hypertension and their family members on hypertension, self-management, and reciprocal family-hypertension impacts to inform future intervention design. DESIGN: We conducted four dyadic focus groups (90-120 minutes) of African American adults with hypertension (i.e. patients) and their family members. We recruited patients (n = 23) and their family members (n = 23) from four African American-serving Christian churches over a period of three months (69.6% female, M age = 60.73 years). Patient-family member dyads were interviewed conjointly (groups ranged from 4 to 6 dyads, each) by facilitators using open-ended questions to elicit perspectives regarding contributors to hypertension, self-management strategies, family influence on self-management, and the impact of hypertension on the family. A grounded theory approach was used for analysis. RESULTS: Participants' responses highlighted themes of societal risk factors and barriers (e.g. racism-related stress worsens blood pressure), influences of African American culture (e.g. culturally-informed diet practices), the patient-physician relationship (e.g. proactive communication is beneficial), family-level influences on health (e.g. family monitoring patients' health behaviors), and patient-level risk factors and self-management strategies (e.g. prayer to cope with stress). Themes reflected a hierarchical, nested, ecological structure such that themes within unique levels of participants' social systems affected, and were affected by, stress, change, or behavior in the other levels. CONCLUSIONS: African American adults with hypertension and their family members described multilevel influences on hypertension and disease self-management, with a strong emphasis on the value of family support. Developing culturally appropriate, family-centered interventions to improve hypertension self-management will be an important next step.

Impact of Adjunctive Pharmacotherapy With Intragastric Balloons for the Treatment of Obesity.

BACKGROUND: We conducted this study to compare the weight loss outcome of intragastric balloons (IGBs) in conjunction with pharmacotherapy vs IGB and intensive lifestyle changes alone. METHODS: This was a multicenter, non-randomized, retrospective study involving 4 academic hospitals. Patients underwent IGB placement with or without concomitant anti-obesity pharmacotherapy. The primary outcome was percent total weight loss (TBWL) after IGB placement at 6 and 12 months. RESULTS: This study included 102 patients, with 23 patients (mean age 46.6 years, 82.6% female) treated with IGB/pharmacotherapy and 79 patients (mean age 46.0 years, 88.6% female) treated with IGB/lifestyle modifications. Patients had a 100% follow-up rate at 6 and 12 months. At 6 months following IGB placement, both groups achieved a similar %TBWL. At 12 months, %TBWL was greater in the IGB/pharmacotherapy group (12.6% ± 1.2 vs 9.7% ± 0.7, P = .04). 65.2% of patients achieved ≥10% TBWL at 12 months in the IGB/pharmacotherapy group, compared to 38.0% in the IGB/lifestyle group (P < .05). The proportion of patients that achieved ≥15% weight loss at 12 months was also significantly different between the IGB/pharmacotherapy and IGB/lifestyle groups (30.4% vs 20.3%, P < .05). DISCUSSION: IGB with concomitant use of pharmacotherapy did not improve weight loss while the IGB was in place compared to IGB and lifestyle changes. However, patients receiving IGB with pharmacotherapy did have greater weight loss and diminished weight regain after balloon removal compared to those receiving just IGB and lifestyle changes.

Obesity and Complications in Mammoplasty: A Retrospective Review in an Obese Patient Population.

With trends of obesity increasing, plastic surgeons are resecting larger weights from larger patients. Published literature has demonstrated the association between body mass index (BMI) and resection weight to postsurgical complications; however, these relationships are unclear in a population that is primarily overweight or obese. Our study examines these relationships to assist plastic surgeons in identifying high-risk patients and discussing preoperative measures to decrease the likelihood of surgical complications. Methods: We performed a retrospective electronic medical record review of a cohort of 182 bilateral reduction mammoplasty procedures performed at a single institution over a four-year period. Patient data were obtained and correlated with postoperative complications. Results: Within our identified patient cohort, 95% were classified as either overweight or obese. Incidence of complications was 51%, with wound dehiscence having the highest incidence of 36.26%. Using a multivariate regression, our analysis found statistical significance between surgical complications and both smoking status and BMI (P = 0.042 and P = 0.025, respectively). Smokers had an increased risk of complications with an odds ratio of 5.165. For every additional 1 kg/m2 increase in BMI, the odds for surgical complication increased by 1.079. In a subanalysis focusing on wound dehiscence, the use of postoperative drains was a protective factor (P = 0.0065). Conclusions: Our study population, with a high average BMI and smoking status, demonstrated a statistically significant increase in postsurgical complications. These findings will help counsel obese patients preoperatively on their increased risk of complications.

Fragment screening at AstraZeneca: developing the next generation biophysics fragment set.

Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.

Microtubules are not required to generate a nascent axon in embryonic spinal neurons in vivo.

Our understanding of the cell behaviours and cytoskeletal requirements of axon formation is largely derived from in vitro models but how these relate to axon formation in vivo is not clear. In vitro, neurons progress through a well-defined multineurite stage to form an axon and both actin and microtubules cooperate to drive the first steps in neurite and axon morphogenesis. However, these steps are not recapitulated in vivo, and it is not clear whether the underlying cell biological mechanisms may differ also. Here, we investigate the mechanisms that regulate axon formation in embryonic zebrafish spinal neurons in vivo. We find microtubule organising centres are located distant from the site of axon initiation, and microtubule plus-ends are not enriched in the axon during axon initiation. Focal F-actin accumulation precedes axon formation, and we find that nocodazole-treated neurons with no detectable microtubules are still able to form nascent axonal protrusions that are approximately 10-µm long, dilated and relatively long-lived. We suggest spinal axon formation in vivo is fundamentally different from axon formation in in vitro models.