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Information on cetacean distribution plays an important role in the identification of suitable boundaries for marine protected areas, but is also crucial for developing management and monitoring programmes. In response to the European ‘Habitats Directive’, a candidate Special Area of Conservation (cSAC) has been established in the Moray Firth, northeast Scotland to protect a small and isolated population of common bottlenose dolphins (Tursiops truncatus). Limited data on the distribution of bottlenose dolphins and on temporal changes in distribution have recently constrained attempts to mitigate against the impacts of new developments upon this population. In response to the need for current information on the distribution of dolphins throughout the cSAC, this study aims to provide data on the distribution of dolphins and other small cetaceans throughout the Moray Firth. Changes in the distribution patterns of dolphins in the inner Moray Firth were examined using data collected between 1990 and 2000. In addition, combined passive acoustic and visual survey techniques were used to determine the distribution of dolphins and harbour porpoises (Phocoena phocoena) on a broader scale across the whole Moray Firth. Dolphin schools were distributed throughout the inner Moray Firth, but there were concentrations of sightings around three deep, narrow channels that were consistent over the ten year study period. Results from surveys across the whole of the Moray Firth showed that all sightings and acoustic detections of dolphins were made within the area of the cSAC. In contrast, porpoise sightings were widely distributed throughout the Moray Firth. The median encounter rate of porpoises across the whole Moray Firth was 1.69 per 100km. Encounter rates of porpoises were similar in the outer Moray Firth and the cSAC. This combination of distribution studies at differing spatial scales provides a valuable tool for monitoring the distribution of animals and identifying important habitats, and the results of this study have directly supported efforts to manage the cSAC.

Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous “neodermis” within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1−/− mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site. Article Highlights Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.

Background: Relative fat mass (RFM) is a novel sex-specific anthropometric equation (based on height and waist measurements) to estimate whole-body fat percentage. Objective: To examine associations of RFM with incident type-2 diabetes (T2D), and to benchmark its performance against body-mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR). Methods: This prospective longitudinal study included data from three Dutch community-based cohorts free of baseline diabetes. First, we examined data from the PREVEND cohort (median age and follow-up duration: 48.0 and 12.5 years, respectively) using Cox regression models. Validation was performed in the Lifelines (median age and follow-up duration: 45.5 and 3.8 years, respectively) and Rotterdam (median age and follow-up duration: 68.0 and 13.9 years, respectively) cohorts. Results: Among 7961 PREVEND participants, 522 (6.6%) developed T2D. In a multivariable model, all adiposity indices were significantly associated with incident T2D (P allBMI: 1.68 (95%CI: 1.57-1.80), HR WC: 1.77 (95% CI: 1.63-1.92) and HR WHR: 1.61 (95%CI: 1.48-1.75)], an equivalent increase in RFM was associated with 119% increased risk [HR: 2.19 (95%CI: 1.96-2.44)]. RFM was associated with incident T2D across all age groups, with the largest effect size in the youngest (

Female patients with chronic pelvic pain (CPP) face complicated healthcare journeys, but narrative perspectives on CPP treatment are lacking.We collected data in English and Spanish from discussion groups and individual interviews with stakeholders around female CPP.A tertiary care center for gynecologic care.Patients with CPP who self-identified as women/female, community health workers (CHWs), and providers who care for women with CPP.We conducted discussion groups with all 3 types of stakeholders and and individual interviews with female patients who have CPP.Patient participants completed condition specific validated questionnaires. De-identified transcripts were coded with NVivo software. We contrasted patient characteristics and codes between CPP patients that did and did not report opioid use in the last 90 days. The mean pain score of patient participants was 6/10 ± 2/10, and 14/47 (28%) reported recent opioid use, without significant differences between patients with and without recent opioid use. Thematic saturation was achieved. Five main themes emerged: the debilitating nature of CPP, emotional impacts of CPP, challenges in CPP healthcare interactions, treatment for CPP, and the value of not feeling alone. Common threads voiced by stakeholders included difficulty discussing chronic pain with others, a sense of inertia in treatment, interest in alternative and less invasive treatments prior to more involved treatments, and the need for individualized, stepwise, integrated treatment plans. Participants agreed that opioids should be used when other treatments fail, but women recently using opioids voiced fewer concerns about addiction and positive experiences with opioid efficacy.These findings amongst female patients with CPP and also amongst CHWs and providers advocate for a move toward patient-centered care, particularly the acknowledgement that every woman experiences pain in a singular way. Furthermore, stakeholders voice a deep need for development of individualized treatment plans.

ObjectivesMalignant mesothelioma is an uncommon cancer associated with asbestos exposure, predominantly occupational. Asbestos has been banned in Australia since 2003 but mesothelioma has a long latency and incident cases continue to present. The Australian Mesothelioma Registry was incepted to collect systematic data about incidence and mortality alongside asbestos exposure.MethodsBenefiting from the Australian national system of cancer notification, all incident cases of mesothelioma in all states and territories are fast-tracked and notified regularly. Notified patients are contacted asking for consent to collect exposure information, initially by postal questionnaire and subsequently by telephone interview. Age-standardised annual incidence rates and mortality rates were calculated. Asbestos exposure was categorised as occupational, non-occupational, neither or, both; and as low, or high, probability of exposure.ResultsMesothelioma incidence appears to have peaked. The age-standardised incidence rates have declined steadily since the early 2000s (peaking in males at 5.9/100 000 and in all-persons at 3.2/100 000), driven by rates in males, who comprise the majority of diagnosed cases. Rates in women have remained fairly stable since that time. Age-standardised mortality rates have followed similar trends. Mesothelioma remains the most common in those aged over 80 years. Nearly all (94%) cases were linked with asbestos exposure (78% occupational in men; 6.8% in women).ConclusionsWith effective control of occupational asbestos use, the decline in age-standardised incidence and death rates has occurred. Incidence rates among women, in whom occupational asbestos exposure is rarely detectable, remain unchanged, pointing to the role of household and /or environmental asbestos exposure.

Aims The FLXibility Post-Approval Study collected data on unselected patients implanted with a WATCHMAN FLX in a commercial clinical setting. Methods and results Patients were implanted with a WATCHMAN FLX per local standard of care, with a subsequent first follow-up visit from 45 to 120 days post-implant and a final follow-up at 1-year post-procedure. A Clinical Event Committee adjudicated all major adverse events and TEE/CT imaging results were adjudicated by a core laboratory. Among 300 patients enrolled at 17 centres in Europe, the mean age was 74.6 ± 8.0 years, mean CHA2DS2-VASc score was 4.3 ± 1.6, and 62.1% were male. The device was successfully implanted in 99.0% (297/300) of patients. The post-implant medication regimen was DAPT for 87.3% (262/300). At first follow-up, core-lab adjudicated complete seal was 88.2% (149/169), 9.5% (16/169) had leak 5 mm leak. At 1 year, 93.3% (280/300) had final follow-up; 60.5% of patients were on a single antiplatelet medication, 21.4% were on DAPT, 5.6% were on direct oral anticoagulation, and 12.1% were not taking any antiplatelet/anticoagulation medication. Adverse event rates through 1 year were: all-cause death 10.8% (32/295); CV/unexplained death 5.1% (15/295); disabling and non-disabling stroke each 1.0% (3/295, all non-fatal); pericardial effusion requiring surgery or pericardiocentesis 1.0% (3/295); and device-related thrombus 2.4% (7/295). Conclusion The WATCHMAN FLX device had excellent procedural success rates, high LAA seal rates, and low rates of thromboembolic events in everyday clinical practice.

Smartphone applications (SPA) now offer the ability to provide accessible in-home monitoring of relevant individual health biomarkers. Previous cross-sectional validations of similar technologies have reported acceptable accuracy with high-grade body composition assessments; this research assessed longitudinal agreement of a novel SPA across a self-managed weight loss intervention of thirty-eight participants (twenty-one males, seventeen females). Estimations of body mass (BM), body fat percentage (BF%), fat-free mass (FFM) and waist circumference (WC) from the SPA were compared with ground truth (GT) measures from a dual-energy X-ray absorptiometry scanner and expert technician measurement. Small mean differences (MD) and standard error of estimate (SEE) were observed between method deltas (ΔBM: MD = 0·12 kg, SEE = 2·82 kg; ΔBF%: MD = 0·06 %, SEE = 1·65 %; ΔFFM: MD = 0·17 kg, SEE = 1·65 kg; ΔWC: MD = 1·16 cm, SEE = 2·52 cm). Concordance correlation coefficient (CCC) assessed longitudinal agreement between the SPA and GT methods, with moderate concordance (CCC: 0·55–0·73) observed for all measures. The novel SPA may not be interchangeable with high-accuracy medical scanning methods yet offers significant benefits in cost, accessibility and user comfort, in conjunction with the ability to monitor body shape and composition estimates over time.

Poly(2-n-propyl-2-oxazine) is better solvated and shows higher backbone flexibility than its oxazoline analogue in dichloromethane, resulting in short distances between chain ends and ultimately increased cyclization efficiency.

HypothesisSelf-assembly of amphipathic styrene maleic acid copolymers with phospholipids in aqueous solution results in the formation of ‘nanodiscs’ containing a planar segment of phospholipid bilayer encapsulated by a polymer belt. Recently, studies have reported that lipids rapidly exchange between both nanodiscs in solution and external sources of lipids. Outstanding questions remain regarding details of polymer-lipid interactions, factors influencing lipid exchange and structural effects of such exchange processes. Here, the dynamic behaviour of nanodiscs is investigated, specifically the role of membrane charge and polymer chemistry.ExperimentsTwo model systems are investigated: fluorescently labelled phospholipid vesicles, and Langmuir monolayers of phospholipids. Using fluorescence spectroscopy and time-resolved neutron reflectometry, the membrane potential, monolayer structure and composition are monitored with respect to time upon polymer and nanodisc interactions.FindingsIn the presence of external lipids, polymer chains embed throughout lipid membranes, the extent of which is governed by the net membrane charge. Nanodiscs stabilised by three different polymers will all exchange lipids and polymer with monolayers to differing extents, related to the properties of the stabilising polymer belt. These results demonstrate the dynamic nature of nanodiscs which interact with the local environment and are likely to deposit both lipids and polymer at all stages of use.

In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time.

Biomaterial-associated infections are one of the major causes of implant failure. These infections result from persistent bacteria that have adhered to the biomaterial surface before, during, or after surgery and have formed a biofilm on the implant's surface. It is estimated that 4 to 10% of implant surfaces are contaminated with bacteria; however, the infection rate can be as high as 30% in intensive care units in developed countries and as high as 45% in developing countries. To date, there is no clinical solution to prevent implant infection without relying on the use of high doses of antibiotics supplied systemically and/or removal of the infected device. In this study, melimine, a chimeric cationic peptide that has been tested in Phase I and II human clinical trials, was immobilized onto the surface of 3D-printed medical-grade polycaprolactone (mPCL) scaffolds via covalent binding and adsorption. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) spectra of melimine-treated surfaces confirmed immobilization of the peptide, as well as its homogeneous distribution throughout the scaffold surface. Amino acid analysis showed that melimine covalent and noncovalent immobilization resulted in a peptide density of ∼156 and ∼533 ng/cm

Ventricular assist device (VAD) driveline exit site infection is a common complication. 3D scaffolds manufactured with highly homogeneous pores via melt electro-writing (MEW) may generate an improved skin-driveline interface which permits cellular in-growth and creates a barrier to prevent bacterial migration along the driveline tissue tunnel. This study investigated skin integration on segments of Heartmate 3 driveline: smooth polyurethane, velour, and on a custom MEW scaffold in a small animal model.Drivelines with surfaces consisting of smooth polyurethane, velour bonded to smooth polyurethane, and smooth polyurethane with a MEW scaffold sleeve were implanted percutaneously in the dorsum of 42 rats. Each rat was implanted with 2 pieces of driveline of 2 cm in length. Skin integration was assessed after 7 and 14 days.Macroscopically, velour and MEW scaffold surfaces were anchored at the driveline-skin interface while smooth polyurethane samples were not attached. The histology analyses showed epidermal migration throughout the thickness of the velour and MEW scaffold groups. Evident tissue growth around single MEW scaffold fibers resulted in full coverage of the pores, while areas of compacted fibers were apparent in the velour group. Tissue ingrowth was significantly higher in the MEW group compared to the velour group after 7 (p0.0001) and 14 days (p0.0001). Marsupialization was observed in the smooth polyurethane samples. Mechanical pull-out forces were similar between velour and MEW scaffold groups at 7 and 14 days (p0.05).Velour and MEW scaffolds promoted epidermal integration while smooth polyurethane drivelines did not. Fine control of MEW scaffold structure production resulted in full cellular coverage and may reduce driveline infection.

© Copyright 2022. Sociedad Meteorológica Estadounidense (AMS). Para obtener permiso para reutilizar cualquier parte de este Trabajo, comuníquese con permisos@ametsoc. org . Cualquier uso del material en este Trabajo que se determine como "uso justo" según la Sección 107 de la Ley de derechos de autor de EE. UU. (17 Código de EE. UU. § 107) o que cumpla las condiciones especificadas en la Sección 108 de la Ley de derechos de autor de EE. ) no requiere el permiso de la AMS. La republicación, la reproducción sistemática, la publicación en forma electrónica, como en un sitio web o en una base de datos de búsqueda, u otros usos de este material, excepto los exentos de la declaración anterior, requieren un permiso por escrito o una licencia de la AMS. Todas las publicaciones periódicas y monográficas de AMS están registradas en el Centro de Autorización de Derechos de Autor (https://www.copyright.com ). Se proporcionan detalles adicionales en la declaración de política de derechos de autor de AMS, disponible en el sitio web de AMS ( https://www.ametsoc.org/PUBSCopyrightPolicy ) ., In 2021, both social and economic activities began to return towards the levels preceding the COVID-19 pandemic for some parts of the globe, with others still experiencing restrictions. Meanwhile, the climate has continued to respond to the ongoing increase in greenhouse gases and resulting warming. La Niña, a phenomenon which tends to depress global temperatures while changing rainfall patterns in many regions, prevailed for all but two months of the year. Despite this, 2021 was one of the six-warmest years on record as measured by global mean surface temperature with an anomaly of between +0.21° and +0.28°C above the 1991–2020 climatology., Lake surface water temperatures from satellite data have been generated within the GloboLakes project funded by the UK National Environment Research Council (NE/J023345/2), with extensions funded by the EU Copernicus Climate Change Service (C3S) programme...

Historical near-surface wind speed (NSWS; ~10 m above the ground) measurements from terrestrial weather stations are crucial for assessing NSWS changes and variability and its implications for various socioeconomic and environmental issues, such as wind energy. However, currently there is no all-Spain gridded NSWS observation product with higher spatial coverage than station-based wind series. A new methodological approach based on image reconstruction using artificial intelligence could help to solve this limitation. We use a partial convolutional neural network (PCNN) and station-based NSWS series from the Spanish Meteorological Agency (AEMET) to create a 0.1º daily gridded wind speed observation product over Spain for 1961-2021. The deep neural network is trained with wind data from the ERA5-Land reanalysis (at 9-km grid-spacing, ECMWF), and a mask where grid points with historical wind observations are identified. Thus, the 0.1º resolution wind distribution grid is treated as the pixel values of an image with the masked grid points being pixels to be reconstructed. The training process allows the PCNN model to learn the physical laws, such as momentum conservation, present as internal relationships between pixels in the reanalysis data. The learned laws were then implemented to estimate the wind speed of the masked grid points. During the training process, the PCNN model predictions are iteratively compared to the reanalysis data and improved according to the error (i.e., MAE or RMSE) between prediction and the original reanalysis data. Once trained, the model is applied to NSWS measurements in the target domain to predict wind at locations with no observations. The gridded NSWS product provides a high-resolution wind speed data for whole Spain that respects the available observations and reliably predict wind speed in unsampled places, which is useful to many applications requiring wind information.

Assessing APOBEC3 gene expression using immunohistochemical and bioinformatic approaches.

APOBEC3 gene expression during senescence and cell cycle progression.

Supplementary Table 1: Overview of data resource. Supplementary Table 2: List of antibodies used for immunofluorescence. Supplementary Table 3: List of antibodies used for western blotting. Supplementary Table 4. List of primers used for PCR.

Generation and characterization of A3A and A3B knockouts in TIIP cells.

A complete list of investigators in the Tracking Non-Small Cell Lung Cancer Evolution through Therapy (TRACERx) Consortium

Correlation between APOBEC3 expression and different measures of CIN.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution.Significance:This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355

This file contains the following supplemental data: Supplemental Table 1. Specific information for MTT cell viability assays with single agents. Supplemental Table 2. Patient characteristics of synovial sarcoma cohort. Supplemental Table 3. Specifications of antibodies used for IHC and Western Blot. Supplemental Table 6. Summary of SS cell line sensitivity to specific tyrosine kinase inhibitors. Supplemental Figure 1. Integrated protein-protein interaction network for all sarcoma subgroups. Supplemental Figure 2. ALK expression and phosphorylation in ES and RMS cell lines. Supplemental Figure 3. Schematic representation of ALK exon 2-17 deletion in Aska-SS cells. Supplemental Figure 4. Deglycosylation of ALK in Aska-SS cells. Supplemental Figure 5. SS cell line sensitivity to foretinib, gefitinib and erlotinib. Supplemental Figure 6. Effects of ALK inhibition on cell cycle and apoptotic markers in Aska-SS cells. Supplemental Figure 7. Effects of ALK inhibition on Aska-SS in vivo tumors. Supplemental Figure 8. ALK, MET and PDGFRA mRNA expression in clinical SS versus other STS samples. Supplemental Figure 9. ALK IHC and patient characteristics of ALK-positive SS tumors. Supplemental Figure 10. Effect of PDGFR and MET inhibition on apoptosis in Yamato-SS cells. Brief sections of Supplementary methods and Supplementary references are included in this file as well.

Supplementary Table S2

Supplementary data - Figure legends and table descriptions

Supplementary Figure S3

Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry–based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALKΔ2–17). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRα phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS. Cancer Res; 77(16); 4279–92. ©2017 AACR.

Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8+ T cells from tumors (not CD4+ T cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8+ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 “normalized” CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers.Significance:NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8+ T-cell exclusion.

Supplementary Figure S4

Protein-protein network analysis

Supplementary Table S1

Purpose: IFN-α is administered to melanoma patients and its endogenous production is essential for immune-mediated tumor recognition. We hypothesized that a reduced capacity for signal transducer and activator of transcription (STAT) 1 activation allows melanoma cells to evade the direct actions of IFN-α.Experimental Design: Tyr701-phosphorylated STAT1 (P-STAT1) was measured by flow cytometry in IFN-α–stimulated human melanoma cell lines, melanoma cells derived from patient tumors, and peripheral blood mononuclear cells (PBMC). Expression of other Janus-activated kinase (Jak)-STAT intermediates (STAT1, STAT2, Jak1, tyrosine kinase 2, IFN-α receptor, STAT3, and STAT5) was evaluated by flow cytometry, immunoblot, or immunohistochemistry.Results: Significant variability in P-STAT1 was observed in human melanoma cell lines following IFN-α treatment (P < 0.05) and IFN-α–induced P-STAT1 correlated with the antiproliferative effects of IFN-α (P = 0.042). Reduced formation of P-STAT1 was not explained by loss of Jak-STAT proteins or enhanced STAT5 signaling as reported previously. Basal levels of P-STAT3 were inversely correlated with IFN-α–induced P-STAT1 in cell lines (P = 0.013). IFN-α–induced formation of P-STAT1 was also variable in melanoma cells derived from patient tumors; however, no relationship between P-STAT3 and IFN-α–induced P-STAT1 was evident. Because IFN-α acts on both tumor and immune cells, we examined the ability of IFN-α to induce P-STAT1 in patient-derived melanoma cells and PBMCs. IFN-α induced significantly lower levels of P-STAT1 in melanoma cells compared with matched PBMCs (P = 0.046). Melanoma cells and human melanocytes required 10-fold higher IFN-α doses to exert P-STAT1 levels comparable with PBMCs.Conclusions: Melanoma cells are variable in their IFN-α responsiveness, and cells of the melanocytic lineage exhibit a lower capacity for IFN-α–induced Jak-STAT signaling compared with immune cells.

Supplementary Figure S6

Supplementary Figure S1

Supplementary Figure S2

Supplementary Data from Melanoma Cells Exhibit Variable Signal Transducer and Activator of Transcription 1 Phosphorylation and a Reduced Response to IFN-α Compared with Immune Effector Cells

Supplementary Figure S5

Intra-hepatic islet transplantation for type-1 diabetes is limited by the need for multiple infusions and poor islet viability post-transplantation. The development of alternative transplantation sites is necessary to improve islet survival, and facilitate monitoring and retrieval. We tested a clinically proven Biodegradable Temporizing Matrix (BTM), a polyurethane-based scaffold, to generate a well vascularized intracutaneous ‘neo-dermis’ within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and facilitated diabetes cure for over 150 days. Further, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is non-toxic for islets. two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days post-BTM implantation. In a pre-clinical porcine skin model, BTM implants created a highly-vascularized intracutaneous site by day 7 post-implantation. When syngeneic neonatal porcine islets were transplanted intracutaneously the islets remained differentiated as insulin producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here we show that BTM facilitates formation of an islet-supportive intracutaneous ‘neo-dermis’ in a porcine pre-clinical model, as an alternative islet transplant site.

Bacteria are continuously exposed to predation from bacteriophages (phages) and, in response, have evolved a broad range of defence systems. These systems can prevent the replication of phages and other mobile genetic elements (MGE). Defence systems are often encoded together in genomic loci defined as “defence islands”, a tendency that has been extensively exploited to identify novel antiphage systems. In the last few years, >100 new antiphage systems have been discovered, and some display homology to components of the immune systems of plants and animals. In many instances, prediction tools have found domains with similar predicted functions present as different combinations within distinct antiphage systems. In this Perspective Article, we review recent reports describing the discovery and the predicted domain composition of several novel antiphage systems. We discuss several examples of similar protein domains adopted by different antiphage systems, including domains of unknown function (DUFs), domains involved in nucleic acid recognition and degradation, and domains involved in NAD+ depletion. We further discuss the potential evolutionary advantages that could have driven the independent acquisition of these domains by different antiphage systems.

Bacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into “defence islands”, providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterise a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 Å, the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localised with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.

Safeners such as metcamifen and benoxacor are widely used in maize to enhance the selectivity of herbicides through the induction of key detoxifying enzymes, notably cytochrome P450 monooxygenases (CYPs). Using a combination of transcriptomics, proteomics, and functional assays, the safener-inducible CYPs responsible for herbicide metabolism in this globally important crop have been identified. A total of 18 CYPs belonging to clans 71, 72, 74, and 86 were safener-induced, with the respective enzymes expressed in yeast and screened for activity toward thiadiazine (bentazon), sulfonylurea (nicosulfuron), and triketone (mesotrione and tembotrione) chemistries. Herbicide metabolism was largely restricted to family CYP81A members from clan 71, notably CYP81A9, CYP81A16, and CYP81A2. Quantitative transcriptomics and proteomics showed that CYP81A9/CYP81A16 were dominant enzymes in safener-treated field maize, whereas only CYP81A9 was determined in sweet corn. The relationship between CYP81A sequence and activities were investigated by splicing CYP81A2 and CP81A9 together as a series of recombinant chimeras. CYP81A9 showed wide ranging activities toward the three herbicide chemistries, while CYP81A2 uniquely hydroxylated bentazon in multiple positions. The plasticity in substrate specificity of CYP81A9 toward multiple herbicides resided in the second quartile of its N terminal half. Further phylogenetic analysis of CYP81A9 showed that the maize enzyme was related to other CYP81As linked to agrochemical metabolism in cereals and wild grasses, suggesting this clan 71 CYP has a unique function in determining herbicide selectivity in arable crops.