Multi-endpoint in vitro toxicological assessment of snus and tobacco-free nicotine pouch extracts.

'Modern' oral tobacco-free nicotine pouches (NPs) are a nicotine containing product similar in appearance and concept to Swedish snus. A three-step approach was taken to analyse the biological effects of NPs and snus extracts in vitro. ToxTracker was used to screen for biomarkers for oxidative stress, cell stress, protein damage and DNA damage. Cytotoxicity, mutagenicity, and genotoxicity were assessed in the following respective assays: Neutral Red Uptake (NRU), Ames and Mouse Lymphoma Assay (MLA). Targeted analysis of phosphorylation signalling and inflammatory markers under non-toxic conditions was used to investigate any potential signalling pathways or inflammatory response. A reference snus (CRP1.1) and four NPs with various flavours and nicotine strengths were assessed. Test article extracts was generated by incubating one pouch in 20 mL of media (specific to each assay) with the inclusion of the pouch material. NP extracts did not induce any cytotoxicity or mutagenic response, genotoxic response was minimal and limited signalling or inflammatory markers were induced. In contrast, CRP1.1 induced a positive response in four toxicological endpoints in the absence of S9: Srxn1 (oxidative stress), Btg2 (cell stress), Ddit3 (protein damage) and Rtkn (DNA damage), and three endpoints in presence of S9: Srxn1, Ddit3 and Rtkn. CRP1.1 was genotoxic when assessed in MLA and activated signalling pathways involved in proliferation and cellular stress and specifically induced phosphorylation of c-JUN, CREB1, p53, p38 MAPK and to a lesser extent AKT1S1, GSK3α/ß, ERK1/2 and RSK1 in a dose-dependent manner. CRP 1.1 extracts resulted in the release of several inflammatory mediators including cytokines IL-1α, IL5, IL6, IL8, IL-1RA, MIF and TNF-ß, receptor IL-2RA, and growth factors FGF-basic, VEGF and M-CSF. In conclusion these assays contribute to the weight of evidence assessment of the potential comparative health risks of NPs and snus.

Determining best practice for technical assessment of hookah surface supply diving equipment during diving fatality investigation.

INTRODUCTION: This study aimed to develop a standard process and checklist for technical investigation of hookah diving equipment and apply it to Tasmanian hookah fatality investigations from the last 25 years. METHODS: A literature search was undertaken to identify technical reports and equipment investigations associated with diving accidents. The information was assimilated to create a process and checklist for specifically assessing the hookah apparatus. The checklist was then applied in a gap analysis of Tasmanian hookah diving fatality technical reports from 1995 to 2019. RESULTS: As no papers specifically describing technical evaluation of hookah equipment were identified, references evaluating scuba equipment were used to create a hookah technical assessment process incorporating unique features of the hookah. Features included: owner responsibility for air quality; maintenance, function; exhaust proximity to air intake; reservoir volume; output non-return valves; line pressure; sufficiency of supply; entanglement; hose severance risk; gas supply failure and hosing attachment to the diver. Seven hookah diving deaths occurred in Tasmania (1995-2019) of which three had documented technical assessment. Gap analysis identified inconsistent structure between reports with variability in the case descriptors. Missing technical data included: overview of the hookah systems; accessories; weights; how the apparatus was worn by the diver; compressor suitability; assessment of hookah function; breathing gas output and exhaust position relative to air intake. CONCLUSIONS: The study demonstrated a need to standardise technical reporting of hookah equipment after diving accidents. The checklist generated may serve as a resource for future hookah assessments and inform strategies for preventing future hookah accidents.

Technical validation of the EMMA capnometer under hyperbaric conditions.

INTRODUCTION: End-tidal carbon dioxide (ETCO2) monitoring is essential for monitoring intubated critical care patients, yet its use in hyperbaric environments can be problematic. We postulated that the EMMA mainstream capnometer may function accurately under hyperbaric conditions. METHODS: Stage 1. The EMMA mainstream capnometer was tested at 101 kPa against a reference side-stream capnometer, Philips IntelliVue M3015B microstream, using 10 customised reference gases of various carbon dioxide (CO2) concentrations (2.47%-8.09%, or 18.5-60.7 mmHg at 101 kPa) in either air or oxygen. Stage 2. The functionality and accuracy of the EMMA capnometer was tested under hyperbaric conditions, 121-281 kPa, using the same test gases. RESULTS: At 101 kPa, the EMMA capnometer measured CO2 at levels lower than expected (mean of differences = -2.5 mmHg (95% CI -2.1 to -2.9, P < 0.001)). The Philips capnometer measured CO2 more closely to expected CO2 (mean of differences = -1.1 mmHg (95% CI -0.69 to -1.4, P < 0.001). Both devices demonstrated a significant linear relationship with expected CO2. The EMMA capnometer functioned up to the maximum test pressure (281 kPa). The device over-read CO2 measurements at pressures > 141 kPa. Although variance increased at pressures in the therapeutic range for hyperbaric treatments, a significant linear relationship between expected and EMMA measured CO2 was demonstrated. The EMMA capnometer tolerated pressures to 281 kPa, but its display was limited to CO2 < 99 mmHg. CONCLUSIONS: This study validated EMMA capnometer function to 281 kPa in the hyperbaric environment. The device over-read CO2 measurements at pressures >141 kPa, however there was a linear relationship between expected and measured CO2. The EMMA capnometer may be clinically useful for monitoring expired CO2 in patients undergoing hyperbaric oxygen treatment.

Safety and performance of intravenous pumps and syringe drivers in hyperbaric environments.

INTRODUCTION: Critically ill patients require continuation of their care when receiving hyperbaric oxygen treatment. This care may be facilitated via portable electrically powered devices such as intravenous (IV) infusion pumps and syringe drivers, which may create risks in the absence of a comprehensive safety evaluation. We reviewed published safety data for IV infusion pumps and powered syringe drivers in hyperbaric environments and compared the evaluation processes to key requirements documented in safety standards and guidelines. METHODS: A systematic literature review was undertaken to identify English language papers published in the last 15 years, describing the safety evaluations of IV pumps and/or syringe drivers for use in hyperbaric environments. Papers were critically assessed in relation to the requirements of international standards and safety recommendations. RESULTS: Eight studies of IV infusion devices were identified. There were deficiencies in the published safety evaluations of IV pumps for hyperbaric use. Despite a simple, published process for evaluating new devices, and available guidelines for fire safety, only two devices had comprehensive safety assessments. Most studies focused only on whether the device functioned normally under pressure and did not consider implosion/explosion risk, fire safety, toxicity, oxygen compatibility or risk of pressure damage. CONCLUSIONS: Intravenous infusion (and other electrically powered) devices require comprehensive assessment before use under hyperbaric conditions. This would be enhanced by a publicly accessible database hosting the risk assessments. Facilities should conduct their own assessments specific to their environment and practices.

Clinical effectiveness of active Alpha-Stim AID versus sham Alpha-Stim AID in major depression in primary care in England (Alpha-Stim-D): a multicentre, parallel group, double-blind, randomised controlled trial.

BACKGROUND: Randomised sham-controlled trials of cranial electrostimulation with the Alpha-Stim Anxiety Insomnia and Depression (AID) device have reported improved anxiety and depression symptoms; however, no adequately powered sham-controlled trials in major depression are available. We investigated whether active Alpha-Stim AID is superior to sham Alpha-Stim AID in terms of clinical effectiveness for depression symptoms in major depression. METHODS: The Alpha-Stim-D trial was a multicentre, parallel group, double-blind, randomised controlled trial, recruiting participants from 25 primary care centres in two regions in England, UK. Eligible participants were aged 16 years or older with a current diagnosis of primary major depression, a score of 10-19 on the nine-item Patient Health Questionnaire, and had been offered or prescribed and reported taking antidepressant medication for at least 6 weeks in the previous 3 months. Main exclusion criteria were contraindications to Alpha-Stim AID device use, having persistent suicidal ideation or self-harm, neurological conditions, a substance use disorder or dependence, an eating disorder, bipolar disorder, or non-affective psychosis, or receiving psychological treatment in the past 3 months. Eligible participants were randomly assigned (1:1, minimised by region, anxiety disorder, and antidepressant use) to 1 h daily use of active (100 µA) or sham Alpha-Stim AID treatment for 8 weeks. Randomisation was via an independent web-based system, with participants, outcome assessors, and data analyst masked to treatment assignment. The primary outcome was change from baseline in score on the 17-item Hamilton Depression Rating Scale (HDRS-17, GRID version) at 16 weeks after randomisation, with participants analysed by intention to treat (ITT; all randomly assigned participants). Safety was assessed in all randomly assigned participants. The trial is registered with the ISRCTN registry (ISRCTN11853110); status completed. FINDINGS: Between Sept 8, 2020, and Jan 14, 2022, 236 eligible participants were randomly assigned to active or sham Alpha-Stim AID (n=118 each). 156 (66%) participants were women, 77 (33%) were men, and three (1%) self-reported as other gender; 200 (85%) were White British or Irish; and the mean age was 38·0 years (SD 15·3; range 16-83). 102 (86%) participants in the active Alpha-Stim AID group and 98 (83%) in the sham group were followed up 16 weeks after randomisation. In the ITT population, mean change in GRID-HDRS-17 at 16 weeks was -5·9 (95% CI -7·1 to -4·8) in the active Alpha-Stim AID group and -6·5 (-7·7 to -5·4) in the sham group (mean change difference -0·6 [95% CI -1·0 to 2·2], p=0·46). Among the 236 participants, 17 adverse events were reported in 17 (7%) participants (nine [8%] participants in the active Alpha-Stim AID group; and eight [7%] participants in the sham group). One serious adverse event of suicidal ideation leading to hospitalisation was reported in the sham group, which was judged to be unrelated to the device. INTERPRETATION: Active Alpha-Stim AID was safe and acceptable, but no more clinically effective than sham Alpha-Stim AID in major depression. FUNDING: National Institute for Health Research Applied Research Collaboration East Midlands and Electromedical Products International.

Efficacy of a Fractionated 1540-nm Erbium Glass Laser in the Treatment of Dermatofibromas.

BACKGROUND: Treatment of dermatofibromas, beyond surgical excision, has remained inadequate and elusive. Nonsurgical treatment options are desired by both patients and physicians. Erbium glass lasers are known for targeting and remodeling the dermis but have not yet been studied for the treatment of dermatofibromas. OBJECTIVE: To evaluate the efficacy and safety of the fractional 1540-nm erbium glass laser for treatment of dermatofibromas. METHODS: Thirty-five patients representing 44 dermatofibromas completed 2 consecutive monthly treatments with a non-ablative, fractional 1540-nm erbium glass laser and were evaluated at 4, 8, and 12 weeks after the initial treatment. Dermatofibromas were evaluated using patient surveys given before and after the treatments. RESULTS: By week 4, patients reported improvement in color and texture of the dermatofibromas. These improvements were durable through week 12. No complications were reported. CONCLUSIONS: This study demonstrates that the fractional 1540-nm erbium glass laser may be used to improve the color and texture of dermatofibromas and achieve positive patient reported outcomes after only 2 treatments. J Drugs Dermatol. 2022;21(11):1201-1205. doi:10.36849/JDD.6287.

Hyperbaric Oxygen for Lower Limb Trauma (HOLLT): an international multi-centre randomised clinical trial.

INTRODUCTION: Hyperbaric oxygen treatment (HBOT) is sometimes used in the management of open fractures and severe soft tissue crush injury, aiming to reduce complications and improve outcomes. METHODS: Patients with open tibial fractures were randomly assigned within 48 hours of injury to receive standard trauma care or standard care plus 12 sessions of HBOT. The primary outcome was the incidence of necrosis or infection or both occurring within 14 days of injury. RESULTS: One-hundred and twenty patients were enrolled. Intention to treat primary outcome occurred in 25/58 HBOT assigned patients and 34/59 controls (43% vs 58%, odds ratio (OR) 0.55, 95% confidence interval (CI) 0.25 to 1.18, P = 0.12). Tissue necrosis occurred in 29% of HBOT patients and 53% of controls (OR 0.35, 95% CI 0.16 to 0.78, P = 0.01). There were fewer late complications in patients receiving HBOT (6/53 vs 18/52, OR 0.22, 95% CI 0.08 to 0.64, P = 0.007) including delayed fracture union (5/53 vs 13/52, OR 0.31, 95% CI 0.10 to 0.95, P = 0.04). Quality of life measures at one and two years were superior in HBOT patients. The mean score difference in short form 36 was 2.90, 95% CI 1.03 to 4.77, P = 0.002, in the short musculoskeletal function assessment (SMFA) was 2.54, 95% CI 0.62 to 4.46, P = 0.01; and in SMFA daily activities was 19.51, 95% CI 0.06 to 21.08, P = 0.05. CONCLUSIONS: In severe lower limb trauma, early HBOT reduces tissue necrosis and the likelihood of long-term complications, and improves functional outcomes. Future research should focus on optimal dosage and whether HBOT has benefits for other injury types.

A randomised controlled trial investigating the clinical and cost-effectiveness of Alpha-Stim AID cranial electrotherapy stimulation (CES) in patients seeking treatment for moderate severity depression in primary care (Alpha-Stim-D Trial).

BACKGROUND: Major depression is the second leading cause of years lost to disability worldwide and is a leading contributor to suicide. However, first-line antidepressants are only fully effective for 33%, and only 40% of those offered psychological treatment attend for two sessions or more. Views gained from patients and primary care professionals are that greater treatment uptake might be achieved if people with depression could be offered alternative and more accessible treatment options. Although there is evidence that the Alpha-Stim Anxiety Insomnia and Depression (AID) device is safe and effective for anxiety and depression symptoms in people with anxiety disorders, there is much less evidence of efficacy in major depression without anxiety. This study investigates the effectiveness of the Alpha-Stim AID device, a cranial electrotherapy stimulation (CES) treatment that people can safely use independently at home. The device provides CES which has been shown to increase alpha oscillatory brain activity, associated with relaxation. METHODS: The aim of this study is to investigate the clinical and cost-effectiveness of Alpha-Stim AID in treatment-seeking patients (aged 16 years upwards) with moderate to moderately severe depressive symptoms in primary care. The study is a multi-centre parallel-group, double-blind, non-commercial, randomised controlled superiority trial. The primary objective of the study is to examine the clinical efficacy of active daily use of 8 weeks of Alpha-Stim AID versus sham Alpha-Stim AID on depression symptoms at 16 weeks (8 weeks after the end of treatment) in people with moderate severity depression. The primary outcome is the 17-item Hamilton Depression Rating Scale at 16 weeks. All trial and treatment procedures are carried out remotely using videoconferencing, telephone and postal delivery considering the COVID-19 pandemic restrictions. DISCUSSION: This study is investigating whether participants using the Alpha-Stim AID device display a reduction in depressive symptoms that can be maintained over 8 weeks post-treatment. The findings will help to determine whether Alpha-Stim AID should be recommended, including being made available in the NHS for patients with depressive symptoms. TRIAL REGISTRATION: ISRTCN ISRCTN11853110 . Registered on 14 August 2020.

Application of ToxTracker for the toxicological assessment of tobacco and nicotine delivery products.

Consumer demands and innovation have led to an increasingly diverse range of nicotine delivery systems, driven by a desire to reduce risk associated with traditional combustible cigarettes. This speed of change provides a mandate for rapid new product assessment. We have used the validated technology ToxTracker®, to assess biomarkers of DNA damage, protein misfolding, oxidative and cellular stress, across the categories of cigarette (1R6F), tobacco heating product (THP 1.4) and electronic cigarette (ePen 3). In addition, we compared the commonly used test matrices for tobacco and nicotine products; whole aerosol aqueous extracts (AqE) and gas vapour phase (GVP), determining their suitability across the product categories. We demonstrated a significant reduction in oxidative stress and cytotoxicity for THP 1.4 over cigarette, further reduced for ePen 3, when assessed by both dilution and nicotine dosimetry. We also identified that while the extraction matrices AqE and GVP from combustible products were equivalent in the induced responses, this was not true of the other category examples, moreover THP 1.4 GVP demonstrates a >50 % reduction in both toxicity and cytotoxicity endpoints over AqE. This indicates that unlike cigarette, the active components or toxicants for THP and electronic cigarette are associated with the aerosol fraction of these categories.

Novel use of poly-L-lactic acid filler for the treatment of facial cutaneous atrophy in patients with connective tissue disease.

BACKGROUND: Patients with connective tissue disease (CTD) often suffer from facial cutaneous defects and resultant facial asymmetry. Unfortunately, these issues have been known to be difficult-to-treat, and concern exists regarding the use of cosmetic procedures in this patient population due to the theoretical risk of disease flare-up or reactivation. Injectable poly-L-lactic acid (PLLA) is one type of filler that has been used to treat skin atrophy in patients with morphea and lupus erythematous panniculitis. However, overall, there is a dearth in literature regarding the safety and efficacy of PLLA filler in patients with CTDs. AIMS: This case series intends to evaluate the safety and efficacy of PLLA filler in treating facial atrophy in patients with CTDs. PATIENTS/METHODS: Three patients underwent various treatment courses involving the use of PLLA filler to treat facial atrophy. RESULTS: Two patients demonstrated significant improvement in facial atrophy following their treatment course. No patient experienced reactivation or exacerbation of their CTD following PLLA injection. CONCLUSION: PLLA filler appears to have good viability as a safe and potentially effective treatment for facial atrophy in patients with CTDs.

The development of an in vitro 3D model of goblet cell hyperplasia using MUC5AC expression and repeated whole aerosol exposures.

Goblet cell hyperplasia and overproduction of airway mucin are characteristic features of the lung epithelium of smokers and COPD patients. Tobacco heating products (THPs) are a potentially less risky alternative to combustible cigarettes, and through continued use solus THPs may reduce smoking-related disease risk. Using the MucilAir™ in vitro lung model, a 6-week feasibility study was conducted investigating the effect of repeated cigarette smoke (1R6F), THP aerosol and air exposure. Tissues were exposed to nicotine-matched whole aerosol doses 3 times/week. Endpoints assessed were dosimetry, tight-junction integrity, cilia beat frequency (CBF) and active area (AA), cytokine secretion and airway mucin MUC5AC expression. Comparison of incubator and air exposed controls indicated exposures did not have a significant effect on the transepithelial electrical resistance (TEER), CBF and AA of the tissues. Cytokine secretion indicated clear differences in secretion patterns in response to 1R6F and THP exposure. 1R6F exposure resulted in a significant decrease in the TEER and AA (p=0.000 and p=0.000, respectively), and an increase in MUC5AC positive cells (p=0.002). Repeated THP exposure did not result in a significant change in MUC5AC positive cells. This study demonstrates repeated cigarette smoke whole aerosol exposure can induce these morphological changes in vitro.

South Pacific Underwater Medicine Society guidelines for cardiovascular risk assessment of divers.

The South Pacific Underwater Medicine Society (SPUMS) diving medical for recreational scuba divers was last reviewed in 2011. From 2011 to 2019, considerable advancements have occurred in cardiovascular risk assessment relevant to divers. The SPUMS 48th (2019) Annual Scientific Meeting theme was cardiovascular risk assessment in diving. The meeting had multiple presentations updating scientific information about assessing cardiovascular risk. These were distilled into a new set of guidelines at the final conference workshop. SPUMS guidelines for medical risk assessment in recreational diving have subsequently been updated and modified including a new Appendix C: Suggested evaluation of the cardiovascular system for divers. The revised evaluation of the cardiovascular system for divers covers the following topics: 1. Background information on the relevance of cardiovascular risk and diving; 2. Defining which divers with cardiovascular problems should not dive, or whom require treatment interventions before further review; 3. Recommended screening procedures (flowchart) for divers aged 45 and over; 4. Assessment of divers with known or symptomatic cardiovascular disease, including guidance on assessing divers with specific diagnoses such as hypertension, atrial fibrillation, cardiac pacemaker, immersion pulmonary oedema, takotsubo cardiomyopathy, hypertrophic cardiomyopathy and persistent (patent) foramen ovale; 5. Additional cardiovascular health questions included in the SPUMS guidelines for medical risk assessment in recreational diving; 6. Updated general cardiovascular medical risk assessment advice; 7. Referencing of relevant literature. The essential elements of this guideline are presented in this paper.

Nutrient Storage in the Perennial Organs of Deciduous Trees and Remobilization in Spring - A Study in Almond (Prunus dulcis) (Mill.) D. A. Webb.

The annual dynamics of whole mature almond tree nutrient remobilization in spring and the accumulation of nutrients in perennial tissues during the year were determined by sequential coring, tissue sampling, nutrient analysis, whole tree excavation and biomass estimation for trees grown under four nitrogen rate treatments 140 kg ha-1 N (N140), 224 kg ha-1 N (N224), 309 kg ha-1 N (N309), and 392 kg ha-1 N (N392) over 2 years. Whole tree perennial organ N content was greatest in dormancy then declined through bud swell, flowering and fruit set, achieving the lowest total whole tree nutrient content of perennial organs by March 12 [12-14 days after full bloom (DAFB)] coincident with 60-70% leaf expansion. During this period no net increment in whole tree N content (annual plus perennial N) was observed indicating that tree demand for N for bud break, flowering, fruit set and leaf out was met by remobilized stored N and that there was no net N uptake from soil. Remobilizable N increased with increasing N application up to N309 and was maximal at 44.4 ± 4 kg ha-1 and 37.5 ± 5.7 kg ha-1 for the optimally fertilized N309 in 2012 and 2013 respectively. Net increases in perennial organ N (stored N) commenced 41 DAFB and continued through full leaf abscission at 249 DAFB. Total annual N increment in perennial organs varied from 25 to 60 kg ha-1 and was strongly influenced by N rate and tree yield. N remobilized from senescing leaves contributed from 11 to 15.5 ± 0.6 kg ha-1 to perennial stored N. Similar patterns of nutrient remobilization and storage were observed for P, K, and S with maximal whole tree perennial storage occurring during dormancy and remobilization of that stored P, K, S to support annual tree demands through to fruit set and 70-100% leaf development. Net annual increment in perennial organ P, K, S commenced 98 DAFB and continued through full leaf abscission at 249 DAFB. Organ specific contribution to remobilizable and stored nutrients changes over the growing season are presented. Details of the pattern of perennial organ nutrient allocation, storage, and remobilization provides a framework for the optimal management of nutrients in almond with relevance for other deciduous tree species.

Intensive fertilizer use increases orchard N cycling and lowers net global warming potential.

Nitrogen (N) fertilizer use has simultaneously increased global food production and N losses, resulting in degradation of water quality and climate pollution. A better understanding of N application rates and crop and environmental response is needed to optimize management of agroecosystems. Here we show an orchard agroecosystem with high N use efficiency promoted substantial gains in carbon (C) storage, thereby lowering net global warming potential (GWP). We conducted a 5-year whole-system analysis comparing reduced (224 kg N ha-1 yr-1) and intensive (309 kg N ha-1 yr-1) fertilizer N rates in a California almond orchard. The intensive rate increased net primary productivity (Mg C ha-1) and significantly increased N productivity (kg N ha-1) and net N mineralization (mg N kg-1 soil d-1). Use of 15N tracers demonstrated short and long-term mechanisms of soil N retention. These low organic matter soils (0.3-0.5%) rapidly immobilized fertilizer nitrate within 36 h of N application and 15N in tree biomass recycled back into soil organic matter over five years. Both fertilizer rates resulted in high crop and total N recovery efficiencies of 90% and 98% for the reduced rate, and 72% and 80% for the intensive rate. However, there was no difference in the proportion of N losses to N inputs due to a significant gain in soil total N (TN) in the intensive rate. Higher soil TN significantly increased net N mineralization and a larger gain in soil organic carbon (SOC) from the intensive rate offset nitrous oxide (N2O) emissions, leading to significantly lower net GWP of -1.64 Mg CO2-eq ha-1 yr-1 compared to -1.22 Mg CO2-eq ha-1 yr-1 for the reduced rate. Our study demonstrates increased N cycling and climate mitigation from intensive fertilizer N use in this orchard agroecosystem, implying a fundamentally different result than seen in conventional annual cropping systems.

Rhinovirus 2A is the key protease responsible for instigating the early block to gene expression in infected cells.

Human rhinoviruses (HRVs) express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study, we have compared both the combined and individual ability of the two proteases to shut down cellular gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different HRV species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

Engineering multi-layered tissue constructs using acoustic levitation.

Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.

Hydration and nanomechanical changes in collagen fibrils bearing advanced glycation end-products.

Accumulation of advanced glycation end-products (AGEs) in biological tissues occurs as a consequence of normal ageing and pathology. Most biological tissues are composed of considerable amounts of collagen, with collagen fibrils being the most abundant form. Collagen fibrils are the smallest discernible structural elements of load-bearing tissues and as such, they are of high biomechanical importance. The low turnover of collagen cause AGEs to accumulate within the collagen fibrils with normal ageing as well as in pathologies. We hypothesized that collagen fibrils bearing AGEs have altered hydration and mechanical properties. To this end, we employed atomic force and Brillouin light scattering microscopy to measure the extent of hydration as well as the transverse elastic properties of collagen fibrils treated with ribose. We find that hydration is different in collagen fibrils bearing AGEs and this is directly related to their mechanical properties. Collagen fibrils treated with ribose showed increased hydration levels and decreased transverse stiffness compared to controlled samples. Our results show that BLS and AFM yield complementary evidence on the effect of hydration on the nanomechanical properties of collagen fibrils.

A 20-year analysis of compressed gas diving-related deaths in Tasmania, Australia.

INTRODUCTION: This study reviews diving deaths that occurred in Tasmanian waters over a 20-year period. METHODS: Detailed analysis was undertaken of deaths that occurred from 01 January 1995 to 31 December 2014. The cases were collated from numerous sources. Utilising a chain of events analysis, factors were identified and assigned to predisposing factors, triggers, disabling agents, disabling injuries and cause of death. These were then scrutinised to ascertain regional variables, remediable factors and linkages which may benefit from targeted risk mitigation strategies. RESULTS: Seventeen deaths were identified across this 20-year period, which included one additional case not previously recorded. All were recreational divers and 15 were male. Five were hookah divers, 12 were scuba divers. Important predisposing factors identified included equipment (condition and maintenance), pre-existing health conditions, diver experience and training. These factors can now be used to promote public health messages for divers. CONCLUSIONS: This 20-year study highlighted regional variations for Tasmanian deaths and presents opportunities for strategies to prevent diving deaths in the future. Of particular concern was the diving practice of 'hookah' diving, which has no governing regulations. The study highlighted the importance of applying a structured methodology such as chain of events analysis to scrutinise diving deaths.

Floodplain soil and its bacterial composition are strongly affected by depth.

We studied bacterial abundance and community structure of five soil cores using high-throughput sequencing of the 16S rRNA gene. Shifts in the soil bacterial composition were more pronounced within a vertical profile than across the landscape. Soil organic carbon (SOC) and nitrogen (N) concentrations decreased exponentially with soil depth and revealed a buried carbon-rich horizon between 0.8 and 1.3 m across all soil cores. This buried horizon was phylogenetically similar to its surrounding subsoils supporting the idea that the type of carbon, not necessarily the amount of carbon was driving the apparent similarities. In contrast to other studies, Nitrospirae was one of our major phyla with relatively high abundances throughout the soil profile except for the surface soil. Although depth is the major driver shaping soil bacterial community structure, positive correlations with SOC and N concentrations, however, were revealed with the bacterial abundance of Acidobacteria, one of the major, and Gemmatimonadetes, one of the minor phyla in our study. Our study showed that bacterial diversity in soils below 2.0 m can be still as high if not higher than in the above laying subsurface soil suggesting that various bacteria throughout the soil profile influence major biogeochemical processes in floodplain soils.