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Plasma membrane repair is a fundamental homeostatic process of eukaryotic cells. Here, we report a new function for the conserved cytoskeletal proteins known as septins in the repair of cells perforated by pore-forming toxins or mechanical disruption. Using a silencing RNA screen, we identified known repair factors (e.g. annexin A2, ANXA2) and novel factors such as septin 7 (SEPT7) that is essential for septin assembly. Upon plasma membrane injury, the septin cytoskeleton is extensively redistributed to form submembranous domains arranged as knob and loop structures containing F-actin, myosin IIA, S100A11, and ANXA2. Formation of these domains is Ca 2+ -dependent and correlates with plasma membrane repair efficiency. Super-resolution microscopy revealed that septins and F-actin form intertwined filaments associated with ANXA2. Depletion of SEPT7 prevented ANXA2 recruitment and formation of submembranous actomyosin domains. However, ANXA2 depletion had no effect on domain formation. Collectively, our data support a novel septin-based mechanism for resealing damaged cells, in which the septin cytoskeleton plays a key structural role in remodeling the plasma membrane by promoting the formation of SEPT/F-actin/myosin IIA/ANXA2/S100A11 repair domains., (© 2024. The Author(s).)

Introduction: Acute hematogenous osteomyelitis may have significant morbidity and mortality if undiagnosed. Because it is uncommon in developed countries and has variable presentations, the patient may undergo several healthcare visits prior to diagnosis., Case Report: We report the case of a 9-year-old male who presented with hip and knee pain with associated fevers and was found to have osteomyelitis and intraosseous abscess in the diaphysis of the right femur. He had multiple emergency department and outpatient visits before the ultimate diagnosis was made. He was treated with irrigation and debridement in addition to intravenous antibiotics., Conclusion: Pediatric acute hematogenous osteomyelitis can have subtle presentations, and this case illustrates some of the difficulties in making the diagnosis. This condition should be considered in the workup of a child with undifferentiated fever, pain, or decrease in mobility., Competing Interests: Conflicts of Interest: By the CPC-EM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. The authors disclosed none.

Context: A quadriceps setting (quad set) exercise is commonly utilized following knee injury, but there is great variation in cues that clinicians provide to patients when performing the exercise., Objectives: To determine if internal, external, or visual cues results in the greatest quadriceps electromyographical (EMG) activity during a quadriceps setting exercise in healthy individuals., Design: Descriptive laboratory study., Setting: University research laboratory., Participants: Thirty healthy individuals volunteered for this study. Participants were given one of five cues in a randomized order: internal cue "tighten your thigh muscles," internal cue "push your knee down," external cue "push into the bolster," external cue "push into the strap," or visual biofeedback using the cue "raise the value on the screen as high as you can.", Main Outcome Measures: Normalized vastus lateralis EMG activity., Results: Both visual biofeedback (83.2±24.9%) and "press into the strap" (76.8±24.4%) produced significantly greater (p< 0.001) EMG activity than the push knee down (53.2±27.0%), tighten thigh (52.7±27.3%), or push into the bolster (50.8±26.3) conditions. There was no significant difference (p= 0.10) between the visual biofeedback and "press into the strap" conditions as well as no significant difference (p> 0.38) between the push knee down, tighten thigh, or push into the bolster conditions., Conclusions: If the clinical aim during a quadriceps setting exercise is to obtain the greatest volitional muscle recruitment, the use of visual biofeedback or pressing into a strap is recommended.

Objectives: To assess the safety and efficacy of an allogeneic adipose-derived mesenchymal stem cell preparation (MAG200) in the treatment of knee osteoarthritis over 12 months., Design: A single-centre, double-blind, ascending dose, randomised controlled trial. 40 participants with moderate knee osteoarthritis were randomised to receive a single intra-articular injection of MAG200 (dose cohorts:10, 20, 50, 100 ​× ​10 6  ​cells) or placebo. Primary objectives were safety and efficacy according to a compound responder analysis of minimal clinically important difference in pain (numerical pain rating scale [NPRS]) and function (Knee Injury and Osteoarthritis Outcome Score - Function in Daily Living subscale [KOOS ADL ]) at month 12. Secondary efficacy outcomes included changes from baseline in patient reported outcome measures and evaluation of disease-modification using quantitative MRI., Results: Treatment was well tolerated with no treatment-related serious adverse events. MAG200 cohorts reported a greater proportion of responders than placebo and demonstrated clinical and statistically significant improvement in pain and clinically relevant improvement in all KOOS subscales. MAG200 demonstrated a reproducible treatment effect over placebo, which was clinically relevant for pain in the 10 ​× ​10 6 dose cohort (mean difference NPRS:-2.25[95%CI:-4.47,-0.03, p ​= ​0.0468]) and for function in the 20 ​× ​10 6 and 100 ​× ​10 6 dose cohorts (mean difference KOOS ADL :10.12[95%CI:-1.51,21.76, p ​= ​0.0863] and 10.81[95%CI:-1.42,23.04, p ​= ​0.0810] respectively). A trend in disease-modification was observed with improvement in total knee cartilage volume in MAG200 10, 20, and 100 ​× ​10 6 dose cohorts, with progression of osteoarthritis in placebo, though this was not statistically significant. No clear dose response was observed., Conclusion: This early-phase study provides supportive safety and efficacy evidence to progress MAG200 to later-stage trial development., Trial Registration: ACTRN12617001095358/ACTRN12621000622808., Competing Interests: JF is the Chief Medical Officer of Magellan Stem Cells Pty Ltd, holds stocks or options in Magellan Stem Cells Pty Ltd and is the Current Chair of the Magellan Stem Cells Medical and Scientific Advisory Board. MC declares no conflicts of interest. JW declares no conflicts of interest. KS is an employee of Magellan Stem Cells Pty Ltd. YW is the recipient of National Health and Medical Research Council of Australia Translating Research into Practice Fellowship (APP1168185). AS declares no conflicts of interest. FMC is the recipient of Investigator Grant from the National Health and Medical Research Council of Australia and reports receipt of institutional payment from Magellan Stem Cells Pty Ltd for undertaking the MRI measurements., (© 2024 Magellan Stem Cells.)

This article is the first qualitative systematic review of studies examining the barriers and facilitators to male sex offenders reintegration from prison or secure care into the community. A search of 16 electronic databases produced 14,218 potential sources, which, after screening, resulted in 79 articles for inclusion. Papers were included if they used qualitative research methods about the barriers, facilitators, perceptions, experiences, and attitudes toward community reintegration from prison or secure care for men convicted of sexual offenses. Included papers were critically appraised and the findings were thematically synthesized. The findings identified that formal and cultural aspects of reintegration, such as probation services, stigmatization, and registration, were the three largest barriers that men faced upon their release, with stability aspects, such as positive relationships, religion, and support groups, being key facilitators to their successful reintegration. The implications for future research, and policy and practice, including prioritizing risk assessment and management, offering appropriate and timely treatment and rehabilitation, educating the community, better access to housing and employment, and services adopting a collaborative approach, are discussed., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Checkpoint inhibition (CPI) is a standard therapeutic approach in metastatic renal cell carcinoma (RCC). However, not all patients respond to CPI, and the immune suppressive characteristics of the RCC tumor microenvironment may contribute to treatment failure. Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) is a transmembrane protein expressed on a subset of myeloid cells with M2-like anti-inflammatory properties that has previously been associated with disease recurrence after nephrectomy and poor outcomes when expressed at high levels. PY314 is a humanized monoclonal antibody targeting TREM2 that depletes tumor-associated macrophages. In this study, the combination of PY314 and pembrolizumab was investigated in patients with CPI-refractory RCC. Eligible patients had clear cell RCC with disease progression on prior CPI either in combination or sequentially with VEGF-TKI. Patients were treated with PY314 10 mg/kg in combination with pembrolizumab 200 mg IV every 21 days. The primary objective was to assess safety and tolerability and secondary objectives included pharmacokinetics and anti-tumor activity by RECIST v1.1. Seventeen patients were enrolled with a median age of 67 years, 82% male, 100% had prior CPI, and 76% had received three or more prior lines of therapy. The combination of PY314 and pembrolizumab demonstrated an acceptable safety profile with 47.1% any grade treatment-related adverse events (AE) (including only 5.9% grade ≥ 3), the most common being fatigue, pyrexia, nausea, and infusion-related reactions. One patient achieved a partial response (6%), and four patients had stable disease (24%) as their best response. The median PFS was 1.4 months (95% CI 1.2- 3.8). The combination of PY314 and pembrolizumab was safe, but the limited anti-tumor effect observed suggests that TREM2 targeting in conjunction with PD-1 blockade may not overcome resistance to prior CPI. Further investigation is warranted to determine if improved efficacy can be achieved in IO-naïve settings. Trial Registration: NCT04691375., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Metastasis remains a major challenge in treating breast cancer. Breast tumors metastasize to organ-specific locations such as the brain, lungs, and bone, but why some organs are favored over others remains unclear. Breast tumors also show heterogeneity, plasticity, and distinct microenvironments. This contributes to treatment failure and relapse. The interaction of breast cancer cells with their metastatic microenvironment has led to the concept that primary breast cancer cells act as seeds, whereas the metastatic tissue microenvironment (TME) is the soil. Improving our understanding of this interaction could lead to better treatment strategies for metastatic breast cancer. Targeted treatments for different subtypes of breast cancers have improved overall patient survival, even with metastasis. However, these targeted treatments are based upon the biology of the primary tumor and often these patients' relapse, after therapy, with metastatic tumors. The advent of immunotherapy allowed the immune system to target metastatic tumors. Unfortunately, immunotherapy has not been as effective in metastatic breast cancer relative to other cancers with metastases, such as melanoma. This review will describe the heterogeneic nature of breast cancer cells and their microenvironments. The distinct properties of metastatic breast cancer cells and their microenvironments that allow interactions, especially in bone and brain metastasis, will also be described. Finally, we will review immunotherapy approaches to treat metastatic breast tumors and discuss future therapeutic approaches to improve treatments for metastatic breast cancer.

Background: Continuous professional development is important to maintain standards of care in the healthcare sector. However, in Lagos, Nigeria, the additional burden of COVID-19 and limited resources may provide separate challenges for physiotherapists' continuous professional development (CPD)., Objectives: To examine the availability and perceived quality of continuous professional development opportunities for physiotherapists working before and during the pandemic in Lagos, Nigeria., Method: A qualitative study was conducted with 10 conveniently sampled physiotherapists, recruited via email. Interviews took place via Zoom, and the video function was utilised. Data were collected via semi-structured interviews using a pilot tested interview, and was transcribed and analysed thematically., Results: The main method of workplace teaching pre-COVID-19 and during COVID-19 was bedside teaching (BT), which most participants received. Pre-COVID-19, the main barrier to receiving teaching was a lack of national guidelines providing specific details on CPD. During COVID-19, the main barrier was the difficulty of increased online teaching instead of teaching on real life patients. The main barriers for non-workplace CPD pre-COVID-19 were a lack of availability of learning opportunities and monetary cost of conferences. This was partially combatted by the increasing trend of online learning events during the pandemic, which increased access to non-workplace learning opportunities., Conclusion: Because of COVID-19, most CPD learning opportunities for physiotherapists in Lagos, Nigeria, were online, increasing overall accessibility. Adequate training to improve utilising online learning resources as well as specific guidelines for workplace physiotherapists CPD in Nigeria should be implemented and promoted to improve confidence and quality of care., Clinical Implications: Key insight into the CPD experiences of physiotherapists currently working in Lagos, Nigeria, which can guide policies and improve clinical outcomes., Competing Interests: The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article., (© 2023. The Authors.)

Alarm signals have evolved to communicate pertinent threats to conspecifics, but heterospecifics may also use alarm calls to obtain social information. In birds, mixed-species flocks are often structured around focal sentinel species, which produce reliable alarm calls that inform eavesdropping heterospecifics about predation risk. Prior research has shown that Neotropical species innately recognize the alarm calls of a Nearctic sentinel species, but it remains unclear how generalizable or consistent such innate signal recognition of alarm-calling species is. We tested for the responses to the alarm calls of a Neotropical sentinel forest bird species, the dusky-throated antshrike ( Thamnomanes ardesiacus ), by naive resident temperate forest birds across three continents during the winter season. At all three sites, we found that approaches to the Neotropical antshrike alarm calls were similarly frequent to the alarm calls of a local parid sentinel species (positive control), while approaches to the antshrike's songs and to non-threatening columbid calls (negative controls) occurred significantly less often. Although we only tested one sentinel species, our findings indicate that temperate forest birds can recognize and adaptively respond globally to a foreign and unfamiliar tropical alarm call, and suggest that some avian alarm calls transcend phylogenetic histories and individual ecological experiences.

Highly sensitive and reproducible analysis of samples containing low amounts of protein is restricted by sample loss and the introduction of contaminants during processing. Here, we report an All-in-One digital microfluidic (DMF) pipeline for proteomic sample reduction, alkylation, digestion, isotopic labeling and analysis. The system features end-to-end automation, with integrated thermal control for digestion, optimized droplet additives for sample manipulation and analysis, and an automated interface to liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Dimethyl labeling was integrated into the pipeline to allow for relative quantification of the trace samples at the nanogram level, and the new pipeline was applied to evaluating cancer cell lines and cancer tissue samples. Several known proteins (including HSP90AB1, HSPB1, LDHA, ENO1, PGK1, KRT18, and AKR1C2) and pathways were observed between model breast cancer cell lines related to hormone response, cell metabolism, and cell morphology. Furthermore, differentially quantified proteins (such as PGS2, UGDH, ASPN, LUM, COEA1, and PRELP) were found in comparisons of healthy and cancer breast tissues, suggesting potential utility of the All-in-One pipeline for the emerging application of proteomic cancer sub-typing. In sum, the All-in-One pipeline represents a powerful new tool for automated proteome processing and analysis, with the potential to be useful for evaluating mass-limited samples for a wide range of applications., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Biofilms serve to house diverse microbial communities, which are responsible for the majority of wastewater constituent degradation and transformation in treatment wetlands (TWs). TW biofilm has been generally conceptualized as a relatively uniform film covering available surfaces. However, no studies attaining direct visual 3D representations of biofilm morphology have been conducted. This study focuses on imaging the morphology of detached, gravel-associated, and rhizospheric (Phalaris arundinacea) biofilms from subsurface TW mesocosms. Images obtained through both traditional light microscopy, environmental scanning electron microscopy (E-SEM) and Wet-SEM revealed that TW biofilms are structurally heterogeneous ranging from corrugated films to clusters of aggregates. Features such as water channels and pores were observed suggesting that pollutant transport inside biofilms is complex, and that the interfacial surface area between water and biofilm is much larger than previously understood. Biofilm thickness generally ranged between 170 and 240 μm, with internal biofilm porosities estimated as 34 ± 10 %, reaching a maximum of 50 %. Internal biofilm matrix pore diameters ranged from 1 to 205.2 μm, with a distribution that favored pores and channels smaller than 10 μm, and a mean equivalent spherical diameter of 8.6 μm. Based on the large variation in pore and channel sizes it is expected that a variety of flow regimes and therefore pollutant dynamics are likely to occur inside TW biofilm matrices. Based on the visual evidence and analysis, a new conceptual model was created to reflect the microscale TW biofilm dynamics and morphology. This new conceptual model will serve to inform future biokinetic modelling, microscale hydrology, microbial community assessment, and pollutant treatment studies., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Communication between parents and dependent offspring is critical not only during provisioning, but also in antipredator contexts. In altricial birds, a top cause of reproductive failure is nest predation, and alarm calls both by parents and chicks can serve to alert others and increase the likelihood of offspring escaping predation. Understanding the factors that determine the strength of parental antipredator responses to different nestling alarm calls can provide insight into parent-offspring recognition. The prothonotary warbler (Protonotaria citrea), a host of the obligate brood parasite, the brown-headed cowbird (Molothrus ater), never rejects cowbird young and raises the parasite together with its own offspring. To determine whether warbler parents learn cowbird nestling alarm calls, we presented experimentally parasitized or non-parasitized parents with playbacks of conspecific warbler, parasitic cowbird, and a harmless heterospecific control, eastern bluebird (Sialis sialis), nestling alarm calls. We recorded the latency to respond and the number of chips given by members of the resident warbler pair. We found that parents were most likely to respond to warbler nestling alarm calls, least likely to respond to bluebird calls, with a statistically intermediate likelihood of responding to cowbird calls. Critically, current and past parasitism status did not affect the likelihood of response to any playback or the number of chips given, however, currently parasitized parents had greater response latencies to playbacks than non-parasitized parents. These results suggest that warbler parents do not learn cowbird alarm calls from breeding experiences and, in turn, that cowbirds may employ a generalized, bet-hedging alarm call., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: This study aims to investigate the efficiency and the geometric as well as the dosimetric benefit of magnetic-resonance guided beam gating for stereotactic treatments in moving organs., Method: Patients treated with MR-guided (MRIdian system) SBRT for lung (n = 10) and liver (n = 10) targets were analyzed. Breath-hold gating was performed based on lesion tracking in sagittal cine MRI images. The target offset from the geometric center of the gating window with and without gating was evaluated. A dose reconstruction workflow based on convolution of these 2D position-probability maps and the daily 3D dose distribution was used to estimate the daily delivered dose including motion. The dose to the clinical target volume (CTV) and to a 2-cm ring structure around the planning target volume were evaluated., Results: The applied gating protocol resulted in a mean (±standard deviation) gating efficiency of 55%±16%. Over all patients, the mean target offset (2D-root-mean-square error) was 8.3 ± 4.3 mm, which reduced to 2.4 ± 0.6 mm during gating. The dose reconstruction showed a mean deviation in CTV coverage (D95) from the static plans of -1.7%±1.8% with gating and -12.0%±8.4% if no gating would have been used. The mean dose (Dmean) in the ring structure, with respect to the static plans, showed mean deviations of -0.1%±0.3% with gating and -1.6%±1.8% without gating., Conclusion: The MRIdian system enables gating based on the inner anatomy and the implemented dose reconstruction workflow demonstrated geometric robust delivery of the planned radiation doses., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: World Health Organisation's data show that low and middle-income countries have a higher prevalence of disabilities. Madagascar is the ninth poorest country in the world. This report aims to analyse the current difficulties faced by physiotherapists and physicians working in the hospital setting, and offer recommendations for how healthcare services can develop in the future., Materials and Methods: Data were collected over the course of 3 months in the form of observational logs, interviews, and questionnaires. Interpreters were used in all interviews, and written questionnaires were translated from English to Malagasy., Results: Thematic analysis of all data collected was completed with key themes emerging: difficult working conditions, including high patient volume and limited staff capacity or treatment space; limited recognition of physiotherapy as a profession, with no autonomy for physiotherapy staff; the low standard of clinical reasoning skills being used in practice; and variable levels of clinical competence, with little evidence of active continuing professional development or appropriate training specific to the needs of clinicians., Conclusions: The current practice of physiotherapy in Madagascar mirrors the challenge in low-income settings. This study highlights the ongoing needs of the profession, including increasing clinical reasoning skills, updating the physiotherapy curriculum, and further development of multidisciplinary teamwork.IMPLICATIONS FOR REHABILITATIONThere are approximately 300 physiotherapists in Madagascar serving 25.5 million people in a low-income country with a high prevalence of disability.Developing the core training skills of physiotherapists in areas such as clinical reasoning skills, improved multidisciplinary team working, and access to research will allow physiotherapists to deliver a higher level of patient-centred care focused on rehabilitation.Upskilling physiotherapists and improving rehabilitation standards will result in improved functioning and quality of life for people with disabilities.Developments in multidisciplinary team communication and working will lead to interprofessional respect, improved quality of work, and a focus on patient-centred care.

Lactobacillus species are prominent inhabitants of the human gastrointestinal tract that contribute to maintaining a balanced microbial environment that positively influences host health. These bacterial populations can be altered through use of probiotic supplements or via dietary changes which in turn affect the host health. Utilizing polyphenolic compounds to selectively stimulate the growth of commensal bacteria can have a positive effect on the host through the production of numerous metabolites that are biologically active. Four Lactobacillus strains were grown in the presence of pomegranate (POM) extract. Two strains, namely, L. acidophilus NCFM and L. rhamnosus GG, are commonly used probiotics, while the other two strains, namely, L. crispatus NCK1351 and L. gasseri NCK1342, exhibit probiotic potential. To compare and contrast the impact of POM on the strains' metabolic capacity, we investigated the growth of the strains with and without the presence of POM and identified their carbohydrate utilization and enzyme activity profiles. To further investigate the differences between strains, an untargeted metabolomic approach was utilized to quantitatively and qualitatively define the metabolite profiles of these strains. Several metabolites were produced significantly and/or exclusively in some of the strains, including mevalonate, glutamine, 5-aminoimidazole-4-carboxamide, phenyllactate, and fumarate. The production of numerous discrete compounds illustrates the unique characteristics of and diversity between strains. Unraveling these differences is essential to understand the probiotic function and help inform strain selection for commercial product formulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chamberlain, O'Flaherty, Cobián and Barrangou.)

Background and Purpose: A potential challenge in single-isocenter multi-lesion lung stereotactic body radiotherapy (SBRT) is that patient positioning is not based on each lesion individually, but on the average position of all lesions. This may lead to larger margins compared to treating with one isocenter per lesion, but increases workflow efficiency. The aim of this study was to investigate whether a single-isocenter technique leads to increased normal lung dose compared to a conventional multiple-isocenters technique., Materials and Methods: A cohort of 15 NSCLC patients with two or three lesions previously treated with SBRT was subjected to treatment planning with a multiple-isocenter technique and a single-isocenter technique. For the latter, two margin approaches were evaluated: (1) identical margins for each internal target volume (ITV), assuming an average registration for all lesions in cone-beam CT (CBCT) positioning verification and (2) a smaller margin for the largest lesion, assuming an optimal registration for that lesion. For all 45 treatment plans, mean lung dose (MLD) and lungs-V 20Gy were evaluated. The study was performed following RATING guidelines., Results: The MLD was 4.9 ± 1.9 Gy (mean ± SD) for multiple-isocenters and 5.4 ± 2.1 Gy and 5.3 ± 2.2 Gy for single-isocenter approach 1 and 2, respectively. V 20Gy was 5.5 ± 3.7%, 5.5 ± 3.2% and 5.4 ± 3.3%. A median [range] increase in MLD of 11.6% [-14.9 - 26.8] was observed when comparing single-isocenter treatment plans to those with multiple isocenters. V 20Gy increased by 0.2 [-3.4 - 1.3] percentage points., Conclusion: A single-isocenter SBRT technique for lung patients with multiple targets results in clinically acceptable increases in normal lung dose., Competing Interests: Conflict of interest statement Dr. Hoogeman reports grants from Elekta, grants from Accuray, grants from Varian, other from Accuray, outside the submitted work. Dr. Tanadini-Lang reports that her husband works at Varian Medical Systems. Dr. van Timmeren, dr. Ehrbar, miss Chamberlain, dr. Mayinger, dr. Andratschke and dr. Guckenberger report no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Purpose of this study is to evaluate plan quality on the MRIdian (Viewray Inc., Oakwood Village, OH, USA) system for head and neck cancer (HNC) through comparison of planning approaches of several centers., Methods: A total of 14 planners using the MRIdian planning system participated in this treatment challenge, centrally organized by ViewRay, for one contoured case of oropharyngeal carcinoma with standard constraints for organs at risk (OAR). Homogeneity, conformity, sparing of OARs, and other parameters were evaluated according to The International Commission on Radiation Units and Measurements (ICRU) recommendations anonymously, and then compared between centers. Differences amongst centers were assessed by means of Wilcoxon test. Each plan had to fulfil hard constraints based on dose-volume histogram (DVH) parameters and delivery time. A plan quality metric (PQM) was evaluated. The PQM was defined as the sum of 16 submetrics characterizing different DVH goals., Results: For most dose parameters the median score of all centers was higher than the threshold that results in an ideal score. Six participants achieved the maximum number of points for the OAR dose parameters, and none had an unacceptable performance on any of the metrics. Each planner was able to achieve all the requirements except for one which exceeded delivery time. The number of segments correlated to improved PQM and inversely correlated to brainstem D 0.1cc and to Planning Target Volume1 (PTV) D 0.1cc . Total planning experience inversely correlated to spinal canal dose., Conclusion: Magnetic Resonance Image (MRI) linac-based planning for HNC is already feasible with good quality. Generally, an increased number of segments and increasing planning experience are able to provide better results regarding planning quality without significantly prolonging overall treatment time., (© 2021. The Author(s).)

Background: Main purpose was to describe procedures and identify challenges in the implementation process of adaptive and non-adaptive MR-guided radiotherapy (MRgRT), especially new risks in workflow due to the new technique. We herein report the single center experience for the implementation of (MRgRT) and present an overview on our treatment practice., Methods: Descriptive statistics were used to summarize clinical and technical characteristics of treatment and patient characteristics including sites treated between April 2019 and end of March 2020 after ethical approval. A risk analysis was performed to identify risks of the online adaptive workflow., Results: A summary of the processes on the MR-Linac including workflows, quality assurance and possible pitfalls is presented. 111 patients with 124 courses were treated during the first year of MR-guided radiotherapy. The most commonly treated site was the abdomen (42% of all treatment courses). 73% of the courses were daily online adapted and a high number of treatment courses (75%) were treated with stereotactic body irradiation. Only 4/382 fractions could not be treated due to a failing online adaptive quality assurance. In the risk analysis for errors, the two risks with the highest risk priority number were both in the contouring category, making it the most critical step in the workflow., Conclusion: Although challenging, establishment of MRgRT as a routinely used technique at our department was successful for all sites and daily o-ART was feasible from the first day on. However, ongoing research and reports will have to inform us on the optimal indications for MRgRT because careful patient selection is necessary as it continues to be a time-consuming treatment technique with restricted availability. After risk analysis, the most critical workflow category was the contouring process, which resembles the need of experienced staff and safety check paths., (© 2021. The Author(s).)

Purpose: To explore the factors affecting access to timely trauma care and rehabilitation in Madagascar., Materials and Methods: A qualitative study based in the outpatient departments of two large rehabilitation centres. Semi-structured interviews and focus groups were conducted with 12 patients or family members and 11 healthcare professionals. Interviews and focus groups were conducted with a local interpreter and were audio-recorded and transcribed. The data were analysed deductively with thematic content analysis, utilising the Health Care Access Barriers model., Results: Participants experienced delays in deciding to seek treatment, accessing healthcare facilities and in receiving appropriate treatment. Cognitive barriers included understanding and awareness of healthcare, structural barriers included distance and transportation to health facilities, financial barriers included affordability of healthcare and difficulty accessing funds., Conclusions: Delays to accessing healthcare may result in increased mortality and disability following trauma, as well as increased financial burden. Addressing the acceptability of services should be a focus for future service development, through training and education schemes. More importantly, improving both physical and financial accessibility of services must be a long-term priority. These findings may help to guide the ongoing development of trauma and rehabilitation pathways in Madagascar.IMPLICATIONS FOR REHABILITATIONDelays in accessing timely trauma care and rehabilitation can lead to complications such as pain, infection, malunion of fractures and loss of function, with devastating financial and social consequences for patients and their families.The acceptability of services is a key barrier to accessing hospital care and may be targeted through training schemes for healthcare professionals and education for the public.Improving the physical and financial accessibility of services in the long-term is paramount.Consideration should be given to these issues in the future development of co-ordinated trauma care and rehabilitation pathways in Madagascar.

The aim of this study was to quantify anatomical changes of parotids and submandibular glands and evaluate potential dosimetric advantages during weekly adaptive MR-guided radiotherapy (MRgRT) for the definitive treatment of head and neck cancer (HNC). The data and plans of 12 patients treated with bilateral intensity-modulated radiotherapy for HNC using MR-linac, with weekly offline adaptations, were prospectively evaluated. The positional and volumetric changes of the salivary glands were analyzed by manual segmentation in weekly MRI images and the dosimetric impact of these anatomical changes on the adapted treatment plans was assessed. The mean volume change in parotid and submandibular gland volume was -31.9% ( p < 0.0001) and -29.7% ( p < 0.0001) after five weeks, respectively. The volume change was significantly correlated with the cumulative dose for the respective gland at the time of volume measurement. Inter-parotid distance changed by -5.4% (6.5 mm) on average after five weeks ( p = 0.0005). The distance became significantly smaller only in the left-right direction. The inter-submandibular gland distance changed by 0.7 mm ( p = 0.38). This study demonstrated significant changes in salivary gland volumes and position following daily MR guidance and weekly plan adaptation. Ongoing clinical trials will provide data on the clinical impact of these changes and novel MR-based adaptation strategies.

Background: MR-guided online adaptive stereotactic body radiation therapy (SBRT) for prostate cancer aims to reduce toxicity by full compensation of interfractional uncertainties. However, the process of online adaptation currently takes approximately 45 min during which intrafractional movements remain unaccounted for. This study aims to analyze the dosimetric benefit of online adaptation and to evaluate its robustness over the duration of one treatment fraction., Methods: Baseline MR-scans at a MR-linear accelerator were acquired for ten healthy male volunteers for generation of mock-prostate SBRT plans with a dose prescription of 5 × 7.25 Gy. On a separate day, online MR-guided adaptation (ViewRay ® MRIdian) was performed, and thereafter MR images were acquired every 15 min for 1 h to assess the stability of the adapted plan., Results: A dosimetric benefit of online MR-guided adaptive re-planning was observed in 90% of volunteers. The median D 95 CTV- and D 95 PTV-coverage was improved from 34.8 to 35.5 Gy and from 30.7 to 34.6 Gy, respectively. Improved target coverage was not associated with higher dose to the organs at risk, most importantly the rectum (median D 1cc rectum baseline plan vs. adapted plan 33.3 Gy vs. 32.3 Gy). The benefit of online adaptation remained stable over 45 min for all volunteers. However, at 60 min, CTV-coverage was below a threshold of 32.5 Gy in 30% of volunteers (30.6 Gy, 32.0 Gy, 32.3 Gy)., Conclusion: The dosimetric benefit of MR-guided online adaptation for prostate SBRT was robust over 45 min in all volunteers. However, intrafractional uncertainties became dosimetrically relevant at 60 min and we therefore recommend verification imaging before delivery of MR-guided online adapted SBRT., (© 2021. The Author(s).)

There is great interest in the development of micromotors which can convert energy to motion in sub-millimeter dimensions. Micromachines take the micromotor concept a step further, comprising complex systems in which multiple components work in concert to effectively realize complex mechanical tasks. Here we introduce light-driven micromotors and micromachines that rely on optoelectronic tweezers (OET). Using a circular micro-gear as a unit component, we demonstrate a range of new functionalities, including a touchless micro-feed-roller that allows the programming of precise three-dimensional particle trajectories, multi-component micro-gear trains that serve as torque- or velocity-amplifiers, and micro-rack-and-pinion systems that serve as microfluidic valves. These sophisticated systems suggest great potential for complex micromachines in the future, for application in microrobotics, micromanipulation, microfluidics, and beyond., (© 2021. The Author(s).)

The optimal approach for magnetic resonance imaging-guided online adaptive radiotherapy is currently unknown and needs to consider patient on-couch time constraints. The aim of this study was to compare two different plan optimization approaches. The comparison was performed in 238 clinically applied online-adapted treatment plans from 55 patients, in which the approach of re-optimization was selected based on the physician's choice. For 33 patients where both optimization approaches were used at least once, the median treatment planning dose metrics of both target and organ at risk differed less than 1%. Therefore, we concluded that beam segment weight optimization was chosen adequately for most patients without compromising plan quality., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: In the emergency department (ED), residents and attendings may have a short-term relationship, such as a single shift. This poses challenges to learner assessment, instructional strategy selection, and provision of substantive feedback. We implemented a process for residents to identify goals for ED shifts; characterized residents' goals; and determined how goal identification affected learning, teaching, and feedback., Methods: This was an observational study in a large, tertiary pediatric ED using mixed methods. Residents were asked to identify learning goals for each shift and were asked postshift if they had identified, accomplished, and/or received feedback on these goals. Goals were categorized by Accreditation Council for Graduate Medical Education Core Competencies. Predictors of goal identification, accomplishment, and receipt of feedback were determined. Residents and attendings were interviewed about their experiences., Results: We collected 306 end-of-shift surveys (74% response rate) and 358 goals and conducted 29 interviews. We found that: 1) Goal setting facilitated perceived learning. Residents identified goals 54% of the time. They accomplished 89% of and received feedback on 76% of goals. 2) Residents' perceived weaknesses, future practice settings, and available patients informed their goals. Most goals mapped to patient care (59%) or medical knowledge (37%) competencies. 3) Goal identification helped attendings determine residents' needs. 4) Ideal goals were specific and achievable. 5) Common barriers were busyness of the ED and difficulty creating goals. Residents were less likely to identify goals (odds ratio [OR] = 0.62, 95% confidence interval [CI] = 0.41 to 0.94) and receive feedback on busy evening shifts (OR = 0.19, 95% CI = 0.10 to 0.37) and were most likely to receive feedback overnight (OR = 3.66, 95% CI = 1.87 to 7.14)., Conclusions: Asking residents to identify goals for ED shifts as an instructional strategy facilitated perceived learning, goal accomplishment, and receipt of feedback. Resident-driven goal identification is a simple and effective instructional strategy that physicians can incorporate into their precepting in the ED., (© 2020 by the Society for Academic Emergency Medicine.)

We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.

The introduction of real-time imaging by magnetic resonance guided linear accelerators (MR-Linacs) enabled adaptive treatments and gating on the tumor position. Different end-to-end tests monitored the accuracy of our MR-Linac during the first year of clinical operation. We report on the stability of these tests covering a static, adaptive and gating workflow. Film measurements showed gamma passing rates of 96.4% ± 3.4% for the static tests (five measurements) and for the two adaptive tests 98.9% and 99.99%, respectively (criterion 2%/2mm). The gated point dose measurements in the breathing phantom were 2.7% lower than in the static phantom., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Purpose: To report the feasibility of magnetic resonance imaging-guided cardiac single fraction radioablation (MRgRA) in a patient with dilated cardiomyopathy and recurrent sustained ventricular tachycardia (VT) leading to electrical storms (ES)., Materials/methods: A workflow to perform Stereotactic Arrhythmia Radioablation (STAR) on a hybrid MR-Linac with real-time tracking and beam-gating was established. Challenges of the MRgRA approach included: (a) the safety of a non-MR compatible cardiac implantable electronic device (CIED) in the MR-Linac field, (b) artefacts caused by the CIED and (c) respiratory motion management with cine-tracking of the moving heart. The specific absorption rate and slew rate of the MR-Linac were within the specifications of a MR-conditional CIED. Phantom measurements showed CIED distortion artefacts of less than 1.5 mm. During MR simulation, tracking could be established on the upper liver to avoid interference with the moving heart and CIED extinction artefacts. Areas of anatomical scarring and critical substrate were identified using invasive three-dimensional electroanatomical mapping of the clinical VT during electrophysiological studies and cardiac MR imaging/computed tomography to build a volumetric target., Results: A 71-year-old male patient with non-ischemic dilated cardiomyopathy and recurrent therapy-refractory sustained VT with repetitive implantable cardioverter-defibrillator (ICD) shocks was treated with a single fraction of 25 Gy @85% isodose, cine-tracking time was 46 min, beam-on time 24 min. 24 h post intervention the patient developed an aggravation of the clinical VT and prolonged ES. VT ceased following high-dose dexamethasone administration after 48 h. After this point, the patient remained without any episodes of sustained ventricular tachyarrhythmia requiring ICD interventions until the last follow-up at three months., Conclusion: Real-time tracking and beam-gating were successfully applied in this first MRgRA to treat sustained VT., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Bevacizumab has promising activity against recurrent glioblastoma (GBM). However, acquired resistance to this agent results in tumor recurrence. We hypothesized that vorinostat, a histone deacetylase (HDAC) inhibitor with anti-angiogenic effects, would prevent acquired resistance to bevacizumab., Methods: This multicenter phase II trial used a Bayesian adaptive design to randomize patients with recurrent GBM to bevacizumab alone or bevacizumab plus vorinostat with the primary endpoint of progression-free survival (PFS) and secondary endpoints of overall survival (OS) and clinical outcomes assessment (MD Anderson Symptom Inventory Brain Tumor module [MDASI-BT]). Eligible patients were adults (≥18 y) with histologically confirmed GBM recurrent after prior radiation therapy, with adequate organ function, KPS ≥60, and no prior bevacizumab or HDAC inhibitors., Results: Ninety patients (bevacizumab + vorinostat: 49, bevacizumab: 41) were enrolled, of whom 74 were evaluable for PFS (bevacizumab + vorinostat: 44, bevacizumab: 30). Median PFS (3.7 vs 3.9 mo, P = 0.94, hazard ratio [HR] 0.63 [95% CI: 0.38, 1.06, P = 0.08]), median OS (7.8 vs 9.3 mo, P = 0.64, HR 0.93 [95% CI: 0.5, 1.6, P = 0.79]) and clinical benefit were similar between the 2 arms. Toxicity (grade ≥3) in 85 evaluable patients included hypertension (n = 37), neurological changes (n = 2), anorexia (n = 2), infections (n = 9), wound dehiscence (n = 2), deep vein thrombosis/pulmonary embolism (n = 2), and colonic perforation (n = 1)., Conclusions: Bevacizumab combined with vorinostat did not yield improvement in PFS or OS or clinical benefit compared with bevacizumab alone or a clinical benefit in adults with recurrent GBM. This trial is the first to test a Bayesian adaptive design with adaptive randomization and Bayesian continuous monitoring in patients with primary brain tumor and demonstrates the feasibility of using complex Bayesian adaptive design in a multicenter setting., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Although required for healing, sleep is often disrupted during hospitalization. Blood pressure (BP) monitoring can be especially disruptive for pediatric inpatients and has few clinical indications. Our aim in this pilot study was to reduce unnecessary overnight BP monitoring and improve sleep for pediatric inpatients., Methods: The intervention in June 2018 involved clinician education sessions and updated electronic health record (EHR) orders that enabled the forgoing of overnight BP checks. The postintervention period from July 2018 to May 2019 examined patient-caregiver surveys as outcome measures. These surveys measured inpatient sleep and overnight disruptions and were adopted from validated surveys: the Patient Sleep Questionnaire, expanded Brief Infant Sleep Questionnaire, and Potential Hospital Sleep Disruptions and Noises Questionnaire. Uptake of new sleep-friendly EHR orders was a process measure. Reported patient care escalations served as a balancing measure., Results: Interrupted time series analysis of EHR orders (n pre = 493; n post = 1472) showed an increase in intercept for the proportion of patients forgoing overnight BP postintervention (+50.7%; 95% confidence interval 41.2% to 60.3%; P < .001) and a subsequent decrease in slope each week (-0.16%; 95% confidence interval -0.32% to -0.01%; P = .037). Statistical process control of surveys (n pre = 263; n post = 131) showed a significant increase in sleep duration for patients older than 2, and nighttime disruptions by clinicians decreased by 19% ( P < .001). Annual estimated cost savings were $15 842.01. No major adverse events in patients forgoing BP were reported., Conclusions: A pilot study combining EHR changes and clinician education safely decreased overnight BP checks, increased pediatric inpatient sleep duration, and reduced nighttime disruptions by clinicians., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2020 by the American Academy of Pediatrics.)

Background: Online adaptive radiotherapy is intended to prevent plan degradation caused by inter-fractional tumor volume and shape changes, but time limitations make online re-planning challenging. The aim of this study was to compare the quality of online-adapted plans to their respective reference treatment plans., Methods: Fifty-two patients treated on a ViewRay MRIdian Linac were included in this retrospective study. In total 238 online-adapted plans were analyzed, which were optimized with either changing of the segment weights (n = 85) or full re-optimization (n = 153). Five different treatment sites were evaluated: prostate, abdomen, liver, lung and pelvis. Dosimetric parameters of gross tumor volume (GTV), planning target volume (PTV), 2 cm ring around the PTV and organs at risk (OARs) were considered. The Wilcoxon signed-rank test was used to assess differences between online-adapted and reference treatment plans, p < 0.05 was considered significant., Results: The average duration of the online adaptation, consisting of contour editing, plan optimization and quality assurance (QA), was 24 ± 6 min. The GTV was slightly larger (average ± SD: 1.9% ± 9.0%) in the adapted plans than in the reference plans (p < 0.001). GTV-D 95% exhibited no significant changes when considering all plans, but GTV-D 2% increased by 0.40% ± 1.5% on average (p < 0.001). There was a very small yet significant decrease in GTV-coverage for the abdomen plans. The ring D mean increased on average by 1.0% ± 3.6% considering all plans (p < 0.001). There was a significant reduction of the dose to the rectum of 4.7% ± 16% on average (p < 0.001) for prostate plans., Conclusions: Dosimetric quality of online-adapted plans was comparable to reference treatment plans and OAR dose was either comparable or decreased, depending on treatment site. However, dose spillage was slightly increased.

Microfluidic methods for studying cell invasion can be subdivided into those in which cells invade into free space and those in which cells invade into hydrogels. The former techniques allow straightforward extraction of subpopulations of cells for RNA sequencing, while the latter preserve key aspects of cell interactions with the extracellular matrix (ECM). Here, we introduce "cell invasion in digital microfluidic microgel systems" (CIMMS), which bridges the gap between them, allowing the stratification of cells on the basis of their invasiveness into hydrogels for RNA sequencing. In initial studies with a breast cancer model, 244 genes were found to be differentially expressed between invading and noninvading cells, including genes correlating with ECM-remodeling, chemokine/cytokine receptors, and G protein transducers. These results suggest that CIMMS will be a valuable tool for probing metastasis as well as the many physiological processes that rely on invasion, such as tissue development, repair, and protection., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The gelatinization temperature (T gel ) of starch increases in the presence of sweeteners due to sweetener-starch intermolecular interactions in the amorphous regions of starch. Different starch botanical sources contain different starch architectures, which may alter sweetener-starch interactions and the effects of sweeteners on T gel s. To document these effects, the T gel s of wheat, potato, waxy corn, dent corn, and 50% and 70% high amylose corn starches were determined in the presence of eleven different sweeteners and varying sweetener concentrations. T gel s of 2:1 sweetener solution:starch slurries were measured using differential scanning calorimetry. The extent of T gel elevation was affected by both starch and sweetener type. T gel s of wheat and dent corn starches increased the most, while T gel s of high amylose corn starches were the least affected. Fructose increased T gel s the least, and isomalt and isomaltulose increased T gel s the most. Overall, starch T gel s increased more with increasing sweetener concentration, molar volume, molecular weight, and number of equatorial and exocyclic hydroxyl groups. Starches containing more short amylopectin chains, fewer amylopectin chains that span through multiple clusters, higher number of building blocks per cluster, and shorter inter-block chain lengths exhibited the largest T gel increases in sweetener solutions, attributed to less stable crystalline regions.

Purpose: This study aims to describe the current provision of trauma rehabilitation in Antananarivo, Madagascar and explore the experiences and perspectives of users of this service in order to inform future research and service development. Material and methods: A qualitative study supplemented by a descriptive service review were applied in the central government hospital in Antananarivo. Registers in the rehabilitation department were analyzed for a 3 month period. Semi-structured interviews were conducted with a convenience sample of users of the rehabilitation service and were subject to thematic analysis. Results: One in six patients treated in the rehabilitation department has survived a traumatic injury, with limb injuries comprising the majority. Analysis of interviews with 10 adult patients, following traumatic injuries to the lower limbs (6), upper limbs (3) or polytrauma (1) generated seven broad themes: health seeking behavior, organization of services, clinical management, costs and financial impact, effect on function and role, psychological impact and societal attitudes. Conclusions: Financial constraints and impact on personal finances pervade the overall picture. The perspectives and experiences elucidated will inform future research into the development of context-specific models of care for people with trauma-related disabilities in Madagascar.Implications for rehabilitationPotentially treatable traumatic injury can have a profound effect on function and societal participation in Madagascar.Interventions to increase access to timely acute trauma care and rehabilitation are needed, and may include different models of financing and innovations to reduce materials costs and length of stay.A structured rehabilitation pathway must be integrated to the earliest stages of trauma care, including scrutinizing the use of prolonged immobilization.Consideration should be given to models of care which are financially and geographically accessible to the population.

Establishing clinically relevant single-cell (SC) transcriptomic workflows from cryopreserved tissue is essential to move this emerging immune monitoring technology from the bench to the bedside. Improper sample preparation leads to detrimental cascades, resulting in loss of precious time, money and finally compromised data. There is an urgent need to establish protocols specifically designed to overcome the inevitable variations in sample quality resulting from uncontrollable factors in a clinical setting. Here, we explore sample preparation techniques relevant to a range of clinically relevant scenarios, where SC gene expression and repertoire analysis are applied to a cryopreserved sample derived from a small amount of blood, with unknown or partially known preservation history. We compare a total of ten cell-counting, viability-improvement, and lymphocyte-enrichment methods to highlight a number of unexpected findings. Trypan blue-based automated counters, typically recommended for single-cell sample quantitation, consistently overestimate viability. Advanced sample clean-up procedures significantly impact total cell yield, while only modestly increasing viability. Finally, while pre-enrichment of B cells from whole peripheral blood mononuclear cells (PBMCs) results in the most reliable BCR repertoire data, comparable T-cell enrichment strategies distort the ratio of CD4+ and CD8+ cells. Furthermore, we provide high-resolution analysis of gene expression and clonotype repertoire of different B cell subtypes. Together these observations provide both qualitative and quantitative sample preparation guidelines that increase the chances of obtaining high-quality single-cell transcriptomic and repertoire data from human PBMCs in a variety of clinical settings.

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about two-fold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure., Competing Interests: The long-term aim of this work is to generate a commercially viable probiotic approach to displace multiresistance plasmids and is associated with University Spinout Plasgene of which CMT is sole director. The non-conjugative pCURE plasmids are protected by US Patent 12/299843 “Plasmid Curing” granted in 2014, owned by University of Birmingham Enterprise Ltd, U.K. The work described in this paper forms the basis of a new US Patent application (US62/879994) owned by University of Birmingham Enterprise Ltd, U.K. As current and former employees of the University of Birmingham CMT, CEM, ASH, JUK, ERS, GSL and MAW declare competing interests. CEM is currently employed by Birmingham Research Park Ltd but has not played a role in the work since moving to this position other than checking the accuracy of the manuscript. Since the mouse data is important for the development of pCURE JI and MK as employees of University of Sydney, Centre for Infectious Disease & Microbiology, Westmead Institute of Medical Research declare competing interests. The patented Mutaflor E. coli Nissle 1917 strain was provided by Ardeypharm GmbH free of charge for scientific purposes and cooperation. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Objective: Rehabilitation services play an important role in optimizing functional ability and societal integration for people with disabilities. The Madagascar Rehabilitation Programme (2011-2013) resulted from a global training partnership and led to 8 doctors achieving a university diploma in rehabilitation medicine. This paper describes a 2014 evaluation of the programme methods, results and learning points., Methods: A combination of qualitative methods was used for the evaluation, based on a Theory of Change model, with informants from Madagascar and the UK., Results: Malagasy trainees and UK volunteers gained new theoretical knowledge and practical skills. For Madagascar, it led to changes in working practice and the formation of a national rehabilitation association. Key to its success was the strong collaboration between Malagasy and UK professionals, with support from the University and Ministry of Health in Madagascar, and the UK partners. Having a clear common vision ensured the programme met the needs of the Malagasy clinicians., Conclusion: Rehabilitation is increasingly recognized as an important focus for international development. Successful rehabilitation training programmes can be achieved at modest costs with global health partnerships. The combination of factors that enabled this programme to be a success is reproducible in other contexts.

Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of "load," "transport," and "deliver," which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell-cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond., Competing Interests: The authors declare no conflict of interest.

Brain metastases (BM) from extracranial cancer are associated with significant morbidity and mortality. Effective local treatment options are stereotactic radiotherapy, including radiosurgery or fractionated external beam radiotherapy, and surgical resection. The use of systemic treatment for intracranial disease control also is improving. BM diagnosis, treatment planning, and follow-up is most often based on contrast-enhanced magnetic resonance imaging (MRI). However, anatomic imaging modalities including standard MRI have limitations in accurately characterizing posttherapeutic reactive changes and treatment response. Molecular imaging techniques such as positron emission tomography (PET) characterize specific metabolic and cellular features of metastases, potentially providing clinically relevant information supplementing anatomic MRI. Here, the Response Assessment in Neuro-Oncology working group provides recommendations for the use of PET imaging in the clinical management of patients with BM based on evidence from studies validated by histology and/or clinical outcome., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To contextualize inpatient sleep duration and disruptions in a general pediatric hospital ward by comparing in-hospital and at-home sleep durations to recommended guidelines and to objectively measure nighttime room entries., Methods: Caregivers of patients four weeks - 18 years of age reported patient sleep duration and disruptions in anonymous surveys. Average at-home and in-hospital sleep durations were compared to National Sleep Foundation recommendations. Objective nighttime traffic was evaluated as the average number of room entries between 11:00pm and 7:00am using GOJO brand hand-hygiene room entry data., Results: Among 246 patients, patients slept less in the hospital than at home with newborn and infant cohorts experiencing 7- and 4-h sleep deficits respectively (Newborn: 787 ± 318 min at home vs. 354 ± 211 min in hospital, p < 0.001; Infants: 703 ± 203 min at home vs. 412 ± 152 min in hospital, p < 0.01). Newborn children also experienced >2 h sleep deficits at home when compared to NSF recommendations (Newborns: 787 ± 318 min at home vs. 930 min recommended, p < 0.05). Objective nighttime traffic measures revealed that hospitalized children experienced 7.3 room entries/night (7.3 ± 0.25 entries). Nighttime traffic was significantly correlated with caregiver-reported nighttime awakenings (Spearman Rank Correlation Coefficient: 0.83, p < 0.001)., Conclusion: Hospitalization is a missed opportunity to improve sleep both in the hospital and at home., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The sport of open water swimming (OWS) has been popularized on a global scale. This population of athletes may experience several unique medical conditions, necessitating a review of medical issues and environmental considerations. Clinicians providing care for OWS athletes should be educated and trained to recognize and manage illnesses and conditions that often occur in an effort to ensure athlete safety. This article focuses on the major medical challenges faced in OWS, including a review of cardiorespiratory conditions and infections, competitor factors, water quality concerns, and risk minimization strategies.

Study Objectives: Sleep is critical to a child's health and well-being, but children are likely to sleep less and be awakened more often during the night in the hospital than at home. To date no studies have compared caregiver, nurse, and physician perspectives of nighttime sleep disruptions in the pediatric general medicine setting. Our aim was to assess caregiver, nurse, and physician perspectives on the most frequent in-hospital disruptors of sleep for pediatric patients. Additionally, we evaluated the degree of agreement of those opinions between the caregivers and medical team., Methods: Caregivers, nurses, and physicians were surveyed using the Potential Hospital Sleep Disruption and Noises Questionnaire (PHSDNQ) regarding their opinions on factors that disrupt sleep. Caregiver responses were collected via a convenience sample of patients hospitalized from February to August 2017 and hospital staff was surveyed once regarding overall perception. The perceived percentage of patients disrupted by each factor was calculated and compared among groups using chi-square tests. Using caregiver rank order based on mean response as the reference gold standard, the absolute differences of nurse and physician rank orders were summed and analyzed using a two-sample test of proportion. In addition, staff was asked knowledge and empowerment questions about how to maximize patient sleep in the hospital and responses were compared using chi-square tests., Results: A total of 162 caregivers, 77 nurses (84% response rate), and 81 physicians (90% response rate) completed surveys. Checking vital signs (50%), nurse/physician interruption (49%), and continuous pulse oximetry (38%) were the three most prevalent disruptors of pediatric inpatient sleep as reported by caregivers. Significant differences were observed between caregiver, nurse, and physician responses for pain, anxiety, alarms, noise, and tests ( P ≤ .001 for all). Both nurse and physician rank orders were discordant when compared to caregivers; there was no significant difference between the two staff groups. When compared to physicians, nurses reported doing more to help children sleep in the hospital (33% versus 94%, P < .001)., Conclusions: Although caregivers report medical interventions such as checking vital signs, nurse/physician interruption, and continuous pulse oximetry as the most frequent disruptors of inpatient pediatric sleep, pediatric staff has poor insight into these disruptions., (© 2018 American Academy of Sleep Medicine.)

Optical micromanipulation has become popular for a wide range of applications. In this work, a new type of optical micromanipulation platform, patterned optoelectronic tweezers (p-OET), is introduced. In p-OET devices, the photoconductive layer (that is continuous in a conventional OET device) is patterned, forming regions in which the electrode layer is locally exposed. It is demonstrated that micropatterns in the photoconductive layer are useful for repelling unwanted particles/cells, and also for keeping selected particles/cells in place after turning off the light source, minimizing light-induced heating. To clarify the physical mechanism behind these effects, systematic simulations are carried out, which indicate the existence of strong nonuniform electric fields at the boundary of micropatterns. The simulations are consistent with experimental observations, which are explored for a wide variety of geometries and conditions. It is proposed that the new technique may be useful for myriad applications in the rapidly growing area of optical micromanipulation., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: To overcome challenges with traditional response assessment in anti-angiogenic agents, the current study uses T1 subtraction maps to quantify volumetric radiographic response in monotherapy with cabozantinib, an orally bioavailable tyrosine kinase inhibitor with activity against vascular endothelial growth factor receptor 2 (VEGFR2), hepatocyte growth factor receptor (MET), and AXL, in an open-label, phase II trial in patients with recurrent glioblastoma (GBM) (NCT00704288)., Methods: A total of 108 patients with adequate imaging data and confirmed recurrent GBM were included in this retrospective study from a phase II multicenter trial of cabozantinib monotherapy (XL184-201) at either 100 mg (N = 87) or 140 mg (N = 21) per day. Contrast enhanced T1-weighted digital subtraction maps were used to define volume of contrast-enhancing tumor at baseline and subsequent follow-up time points. Volumetric radiographic response (>65% reduction in contrast-enhancing tumor volume from pretreatment baseline tumor volume sustained for more than 4 wk) was tested as an independent predictor of overall survival (OS)., Results: Volumetric response rate for all therapeutic doses was 38.9% (41.4% and 28.6% for 100 mg and 140 mg doses, respectively). A log-linear association between baseline tumor volume and OS (P = 0.0006) and a linear correlation between initial change in tumor volume and OS (P = 0.0256) were observed. A significant difference in OS was observed between responders (median OS = 20.6 mo) and nonresponders (median OS = 8.0 mo) (hazard ratio [HR] = 0.3050, P < 0.0001). Multivariable analyses showed that continuous measures of baseline tumor volume (HR = 1.0233, P < 0.0001) and volumetric response (HR = 0.2240, P < 0.0001) were independent predictors of OS., Conclusions: T1 subtraction maps provide value in determining response in recurrent GBM treated with cabozantinib and correlated with survival benefit.

Importance: Treatment options for patients with disease progression after treatment with trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1) are limited. Tucatinib is an oral, potent, human epidermal growth factor receptor 2 (HER2)-specific tyrosine kinase inhibitor (TKI) being developed as a novel treatment for ERBB2/HER2-positive breast cancer., Objective: To determine the maximum tolerated dosage of tucatinib in combination with T-DM1 in the treatment of patients with ERBB2/HER2-positive metastatic breast cancer with and without brain metastases., Design, Setting, and Participants: In this phase 1b open-label, multicenter, clinical trial, 57 participants enrolled between January 22, 2014, and June 22, 2015, were 18 years of age or older with ERBB2/HER2-positive metastatic breast cancer previously treated with trastuzumab and a taxane. Data were analyzed between January and March 2018., Interventions: Tucatinib 300 mg or 350 mg administered orally twice per day for 21 days and T-DM1 3.6 mg/kg administered intravenously once every 21 days., Main Outcomes and Measures: Safety assessments, pharmacokinetics, and response were assessed using RECIST 1.1 every 2 cycles for 6 cycles, followed by every 3 cycles., Results: Fifty-seven T-DM1-naive patients (median [IQR] 51 [44.0-63.0] years of age) who had undergone a median of 2 earlier HER2 therapies (range, 1-3) were treated. The tucatinib maximum tolerated dosage was determined to be 300 mg administered twice per day with dose-limiting toxic reactions seen at 350 mg twice per day. Pharmacokinetic analysis showed that there was no drug-drug interaction with T-DM1. Adverse events seen among the 50 patients treated at the maximum tolerated dosage regardless of causality included nausea (36 patients; 72%), diarrhea (30 patients; 60%), fatigue (28 patients; 56%), epistaxis (22 patients; 44%), headache (22 patients; 44%), vomiting (21 patients; 42%), constipation (21 patients; 42%), and decreased appetite (20 patients; 40%); the majority of adverse events were grade 1 or 2. Tucatinib-related toxic reactions that were grade 3 and above included thrombocytopenia (7 patients; 14%) and hepatic transaminitis (6 patients; 12%)., Conclusions and Relevance: In this study, tucatinib in combination with T-DM1 appeared to have acceptable toxicity and to show preliminary antitumor activity among heavily pretreated patients with ERBB2/HER2-positive metastatic breast cancer with and without brain metastases., Trial Registration: ClinicalTrials.gov Identifier: NCT01983501.

Background: Tucatinib is a potent and selective oral HER2 tyrosine kinase inhibitor, with the potential to provide a well tolerated new treatment option for patients whose disease has progressed on currently available therapies. We aimed to determine the recommended phase 2 dose, safety, pharmacokinetics, and preliminary activity of tucatinib in combination with capecitabine or trastuzumab in patients with HER2-positive breast cancer with or without brain metastases., Methods: In this non-randomised, open-label, phase 1b trial done in five sites in the USA, we recruited patients aged 18 years or older with HER2-positive progressive breast cancer who had been previously treated with trastuzumab, pertuzumab, and trastuzumab emtansine. Eligible patients required HER2-positivity assessed locally, evaluable lesions as defined per Response Evaluation Criteria in Solid Tumors, version 1.1, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Tucatinib was administered twice a day in conjunction with capecitabine 1000 mg/m 2 orally twice a day for 14 days of a 21-day cycle, trastuzumab 6 mg/kg intravenously once every 21 days, or both. A modified 3 + 3 dose-escalation design was used to determine the recommended phase 2 dose, starting with tucatinib in combination with capecitabine or trastuzumab, and subsequently evaluating the triplet combination. The primary endpoint was to establish the maximum tolerated dose and recommended phase 2 dose of tucatinib, evaluated by toxicity assessments. Efficacy was assessed in all patients by contrast CT of the body. Analyses included all patients who had received at least one dose of study treatment. The study is registered with ClinicalTrials.gov, number NCT02025192., Findings: Between Jan 15, 2014, and Dec 15, 2015, 60 patients were enrolled and treated. The current report is from mature data as of June 30, 2017. The tucatinib recommended phase 2 dose was determined to be 300 mg orally twice a day, equivalent to single-agent maximum tolerated dose. Pharmacokinetic analysis showed that there was no drug-drug interaction with capecitabine. Adverse events seen at the recommended phase 2 dose regardless of causality, grade, and treatment group included diarrhoea (35 [67%] of 52 patients), nausea (31 [60%] patients), palmar-plantar erythrodysaesthesia syndrome (23 [44%] patients), fatigue (20 [38%] patients), and vomiting (20 [38%] patients). In all patients, treatment-related toxicities of grade 3 and worse included fatigue (five [8%] patients), diarrhoea (four [7%] patients), and palmar-plantar erythrodysaesthesia (four [7%] patients). No treatment-related deaths were reported. The proportion of patients with measurable disease achieving objective response was 83% (five of six patients) in the combination of tucatinib with capecitabine, 40% (six of 15 patients) in the combination of tucatinib with trastuzumab, and 61% (14 of 23 patients) in the combination of tucatinib with both capecitabine and trastuzumab., Interpretation: Tucatinib in combination with capecitabine and trastuzumab had acceptable toxicity and showed preliminary anti-tumour activity. Validation of the current study results will be determined in the double-blinded randomised study, HER2CLIMB (ONT-380-206; NCT02614794)., Funding: Cascadian Therapeutics, a wholly owned subsidiary of Seattle Genetics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The early part of the last deglaciation is characterised by a ~40 ppm atmospheric CO 2 rise occurring in two abrupt phases. The underlying mechanisms driving these increases remain a subject of intense debate. Here, we successfully reproduce changes in CO 2 , δ 13 C and Δ 14 C as recorded by paleo-records during Heinrich stadial 1 (HS1). We show that HS1 CO 2 increase can be explained by enhanced Southern Ocean upwelling of carbon-rich Pacific deep and intermediate waters, resulting from intensified Southern Ocean convection and Southern Hemisphere (SH) westerlies. While enhanced Antarctic Bottom Water formation leads to a millennial CO 2 outgassing, intensified SH westerlies induce a multi-decadal atmospheric CO 2 rise. A strengthening of SH westerlies in a global eddy-permitting ocean model further supports a multi-decadal CO 2 outgassing from the Southern Ocean. Our results highlight the crucial role of SH westerlies in the global climate and carbon cycle system with important implications for future climate projections.