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.

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.

Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes.

AIMS/HYPOTHESIS: Improved biomarkers are acutely needed for the detection of developing type 1 diabetes, prior to critical loss of beta cell mass. We previously demonstrated that elevated beta cell microRNA 21-5p (miR-21-5p) in rodent and human models of type 1 diabetes increased beta cell apoptosis. We hypothesised that the inflammatory milieu of developing diabetes may also increase miR-21-5p in beta cell extracellular vesicle (EV) cargo and that circulating EV miR-21-5p would be increased during type 1 diabetes development. METHODS: MIN6 and EndoC-ßH1 beta cell lines and human islets were treated with IL-1ß, IFN-γ and TNF-α to mimic the inflammatory milieu of early type 1 diabetes. Serum was collected weekly from 8-week-old female NOD mice until diabetes onset. Sera from a cross-section of 19 children at the time of type 1 diabetes diagnosis and 16 healthy children were also analysed. EVs were isolated from cell culture media or serum using sequential ultracentrifugation or ExoQuick precipitation and EV miRNAs were assayed. RESULTS: Cytokine treatment in beta cell lines and human islets resulted in a 1.5- to threefold increase in miR-21-5p. However, corresponding EVs were further enriched for this miRNA, with a three- to sixfold EV miR-21-5p increase in response to cytokine treatment. This difference was only partially reduced by pre-treatment of beta cells with Z-VAD-FMK to inhibit cytokine-induced caspase activity. Nanoparticle tracking analysis showed cytokines to have no effect on the number of EVs, implicating specific changes within EV cargo as being responsible for the increase in beta cell EV miR-21-5p. Sequential ultracentrifugation to separate EVs by size suggested that this effect was mostly due to cytokine-induced increases in exosome miR-21-5p. Longitudinal serum collections from NOD mice showed that EVs displayed progressive increases in miR-21-5p beginning 3 weeks prior to diabetes onset. To validate the relevance to human diabetes, we assayed serum from children with new-onset type 1 diabetes compared with healthy children. While total serum miR-21-5p and total serum EVs were reduced in diabetic participants, serum EV miR-21-5p was increased threefold compared with non-diabetic individuals. By contrast, both serum and EV miR-375-5p were increased in parallel among diabetic participants. CONCLUSIONS/INTERPRETATION: We propose that circulating EV miR-21-5p may be a promising marker of developing type 1 diabetes. Additionally, our findings highlight that, for certain miRNAs, total circulating miRNA levels are distinct from circulating EV miRNA content.

Re-purposing of domestic steam disinfectors within the hospital-at-home setting.

BACKGROUND: Vulnerable patients being cared for in hospital-at-home settings require safe disinfection of their medical devices, including nebulisers and other respiratory equipment. The scale of patients now being cared for in hospital-at-home settings as a result of COVID19 places huge pressure on hospital central sterile services departments (CSSDs) to provide consumable items to safely support such patients' care. This places new importance on the disinfection of mundane objects, including crockery, cutlery and frequently touched objects in the home environment. This study examined temperature performance of steam disinfectors and the consequences of potential operator misuse on the survival of 62 bacteria and yeast organisms. METHODS: Thermal performance of steam disinfectors was evaluated using calibrated thermocouple probes in multiple permutations of device usage with 62 test organisms. RESULTS: Thermocouple data demonstrated disinfection A0 values of 6000 (upper layer) and 60 (lower layer). Steam disinfection of baby bottles had a thermal lethality of at least A0 = 600. Variation in disinfector temperatures were noted, depending on the geometric location of thermocouples. Additional notable temperature reductions occurred with device underfilling with suboptimal water volumes. Steam disinfection eradicated all 62 non-spore-forming Gram-positive, Gram-negative and yeast organisms tested and eradicated all organisms in the inner teat space of contaminated babies' dummies, rendering safe steam disinfection of babies' dummies. CONCLUSION: Domestic steam disinfection offers an inexpensive, simple, versatile and widely available technology for the elimination of common non-spore-forming nosocomial pathogens and safe disinfection of medical devices, fomites and other mundane objects within the hospital-at-home scenario, thereby enhancing patient safety.

Protrusion-Mediated Signaling Regulates Patterning of the Developing Nervous System.

During brain development, the tissue pattern and specification are the foundation of neuronal circuit formation. Contact-mediated lateral inhibition is well known to play an important role in determining cell fate decisions in the nervous system by either regulating tissue boundary formation or the classical salt-and-pepper pattern of differentiation that results from direct neighboring cell contacts. In many systems, however, such as the Drosophila notum, Drosophila wing, zebrafish pigmented cells, and zebrafish spinal cord, the differentiation pattern occurs at multiple-cell diameter distances. In this review, we discuss the evidence and characteristics of long-distance patterning mechanisms mediated by cellular protrusions. In the nervous system, cellular protrusions deliver the Notch ligand Delta at long range to prevent cells from differentiating in their vicinity. By temporal control of protrusive activity, this mechanism can pattern differentiation in both space and time.

Antimicrobial resistance (AMR) and marine plastics: Can food packaging litter act as a dispersal mechanism for AMR in oceanic environments?

This study examined the carriage of antibiotic resistance in bacteria isolated from Food-related Marine Macroplastic Litter (FRMMPL) around the coastline of Northern Ireland. FRMMPL was collected from 18 coastal sites during November/December 2018 and the bacteria from the surface of the plastic examined for their susceptibility to 10 common human antibiotics. Ten bacterial genera and 13 species were identified from the plastic materials. Bacteria isolated from plastic material were most resistant to the beta-lactam antibiotics (ampicillin, ceftazidime and cefpodoxime) (98.1% resistant) and least resistant to the tetracycline group, minocycline (16.1% resistant). This study is significant as it highlights a new potential route of dispersal of such antibiotic-resistance in the environment, which may act as carriers of such bacteria by introducing them into new marine ecosystems, as well as potential pathways having impacts on animal and human health, until their final interaction with the human foodchain.

Importance of Nebulizer Drying for Patients With Cystic Fibrosis.

BACKGROUND: Nebulizer therapy is an important treatment component for patients with cystic fibrosis (CF). Nebulizer manufacturers' guidelines advocate thorough nebulizer drying after washing. The aim of this study, therefore, was to examine the microbiology associated with nebulizer drying, particularly related to Pseudomonas control, and to examine microbiologically non-adherence to the recommended drying procedures. METHODS: Four aspects of nebulizer drying were examined in 3 common nebulizers, including examination of the drying profile, improvement to the drying profile of assembled nebulizers, survival of Pseudomonas aeruginosa in tap water and in tap water plus 0.5% (v/v) dishwashing detergent, and the effect of drying of P. aeruginosa in tap water and tap water plus residual sputum (1%v/v, 10%v/v). Microbiologic examination was performed by using P. aeruginosa (5 clinical CF strains plus 1 National Collection of Type Cultures Reference strain). RESULTS: There were differences in the time to complete dryness between disassembled and fully assembled nebulizers. Vigorous repeated shaking was unable to drive off all residual water on assembled nebulizers. P. aeruginosa counts did not decrease significantly in either tap water or in tap water plus detergent after 24 h storage at ambient temperature. In contrast, all Pseudomonas organisms were killed when nebulizers were dried for 24 h, even when contaminated with 1% and 10% sputum. Dishwashing detergent did not demonstrate any antibacterial activity. CONCLUSIONS: This study demonstrated that nebulizer drying, if applied properly, had the ability to reduce counts of P. aeruginosa to non-detectable levels. Equally, this study showed that, if the device was not dried thoroughly and moisture remained, then the device was able to support the survival of P. aeruginosa at high numbers, which constituted an infection risk to the patient with CF. This information may help educate and inform the patient with CF about the importance of proper nebulizer drying for Pseudomonas control to improve patient awareness and safety.

Steam disinfection of toothbrushes from patients with cystic fibrosis: Evidence-based recommendations.

BACKGROUND: Patients with cystic fibrosis have increased morbidity/mortality due to chronic respiratory infections, which primarily originate from the environment. Infection prevention and control emphasize the importance of cleaning and disinfection of respiratory devices, however, there is a paucity of guidance on toothbrush hygiene, which have been shown to be a source of cystic fibrosis (CF) pathogens. METHODS: This study examined steam disinfection of toothbrushes contaminated with clinically significant CF isolates (n = 80; Gram positive = 33; Gram negative = 32, and non-tuberculous mycobacteria = 6) and yeasts (n = 9), as well as oral streptococci (n = 26) and environmental Pseudomonas aeruginosa (n = 12). RESULTS: Steam disinfection eradicated all organisms tested, as well as all organisms in CF sputum applied to toothbrushes. CONCLUSIONS: Steam disinfection offers a relatively simple, cheap and available method of eliminating non-spore-forming CF pathogens on toothbrushes. Toothbrushes should be thoroughly rinsed after each use before steam disinfection, to remove plaque, epithelial cells, and residual toothpaste. Toothbrushes should be steam disinfected after each use employing a baby bottle steam disinfector, adhering to manufacturers' operating instructions and stored in the disinfector until next used within 12 to 24 hours. Toothbrushes should be replaced every 3 to 4 months, or sooner if the bristles look worn out, as well as every time a pulmonary exacerbation occurs or every time the patient is treated for a pulmonary/throat infection. Steam disinfection of toothbrushes is crucial when the patient is undergoing eradication regimes for P. aeruginosa and methicillin-resistant Staphylococcus aureus, so that the patient does not become reinfected from this source, thereby aiding eradication and enhancing patient safety.

Antimicrobial properties of basidiomycota macrofungi to Mycobacterium abscessus isolated from patients with cystic fibrosis.

BACKGROUND: Antimicrobial resistance (AMR) has now emerged as a global public health crisis. Of particular concern is AMR associated with the genus Mycobacterium, including Mycobacterium tuberculosis and the nontuberculous mycobacteria (NTM). Emergence of the NTM, in particular Mycobacterium abscessus, in patients with cystic fibrosis (CF) represents both a diagnostic and a treatment dilemma. Such resistance drives the need to investigate novel sources of antimicrobials. Medicinal fungi have a well-documented history of use in traditional oriental therapies. Not only is this an ancient practice, but also still today, medical practice in Japan, China, Korea, and other Asian countries continue to rely on fungal-derived antibiotics. A study was, therefore, undertaken to examine the antimicrobial activity of 23 native macrofungal (mushrooms/toadstools) taxa, collected from woodlands in Northern Ireland against six clinical (CF) isolates of M. abscessus, as well as M. abscessus National Collection of Type Cultures (NCTC) Reference strain (NCTC 13031). METHODS: Free-growing saprophytic and mycorrhizal macrofungi (n = 23) belonging to the phylum Basidiomycota were collected and were definitively identified employing Polymerase Chain reaction/ITS DNA sequencing. Macrofungal tissues were freeze-dried and reconstituted before employment in antibiotic susceptibility studies. RESULTS: All macrofungi examined showed varying inhibition of the M. abscessus isolates examined with the exception Russula nigricans. The macrofungi displaying maximum antimycobacterial activity against the clinical isolates were (in descending order) M. giganteus (33.6 mg/ml), Hygrocybe nigrescens (38.5 mg/ml) and Hypholoma fasciculare (25.3 mg/ml). CONCLUSION: Macrofungi may represent a source of novel antimicrobials against M. abscessus, which have not yet been fully explored nor exploited clinically. This is the first report describing the antimycobacterial properties of extracts of M. giganteus against M. abscessus. Further work is now required to identify the constituents and mode of the inhibitory action of these macrofungi against the M. abscessus. Given the gravity of AMR in the NTMs, particularly M. abscessus and the clinical treatment dilemmas that such AMR present, antibiotic drug discovery efforts should now focus on investigating and developing antibacterial compounds from macrofungi, particularly M. giganteus, where there are no or limited current treatment options.

Basal Protrusions Mediate Spatiotemporal Patterns of Spinal Neuron Differentiation.

During early spinal cord development, neurons of particular subtypes differentiate with a sparse periodic pattern while later neurons differentiate in the intervening space to eventually produce continuous columns of similar neurons. The mechanisms that regulate this spatiotemporal pattern are unknown. In vivo imaging in zebrafish reveals that differentiating spinal neurons transiently extend two long protrusions along the basal surface of the spinal cord before axon initiation. These protrusions express Delta protein, consistent with the hypothesis they influence Notch signaling at a distance of several cell diameters. Experimental reduction of Laminin expression leads to smaller protrusions and shorter distances between differentiating neurons. The experimental data and a theoretical model support the proposal that neuronal differentiation pattern is regulated by transient basal protrusions that deliver temporally controlled lateral inhibition mediated at a distance. This work uncovers a stereotyped protrusive activity of newborn neurons that organize long-distance spatiotemporal patterning of differentiation.

Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.

We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms.