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Abstract The effects of the COVID-19 pandemic on comprehensive maternal deaths in Brazil have not been fully explored. Using publicly available data from the Brazilian Mortality Information (SIM) and Information System on Live Births (SINASC) databases, we used two complementary forecasting models to predict estimates of maternal mortality ratios using maternal deaths (MMR) and comprehensive maternal deaths (MMRc) in the years 2020 and 2021 based on data from 2008 to 2019. We calculated national and regional standardized mortality ratio estimates for maternal deaths (SMR) and comprehensive maternal deaths (SMRc) for 2020 and 2021. The observed MMRc in 2021 was more than double the predicted MMRc based on the Holt-Winters and autoregressive integrated moving average models (127.12 versus 60.89 and 59.12 per 100,000 live births, respectively). We found persisting sub-national variation in comprehensive maternal mortality: SMRc ranged from 1.74 (95% confidence interval [CI] 1.64, 1.86) in the Northeast to 2.70 (95% CI 2.45, 2.96) in the South in 2021. The observed national estimates for comprehensive maternal deaths in 2021 were the highest in Brazil in the past three decades. Increased resources for prenatal care, maternal health, and postpartum care may be needed to reverse the national trend in comprehensive maternal deaths.

Objectives: Rates of maternal syphilis have increased five-fold in Brazil in the past decade. While penicillin remains the only appropriate treatment for maternal syphilis, we hypothesized that low non-treponemal titers (

Sexual and gender minority (SGM) and racial or ethnic minority youth at-risk for or living with HIV may have higher risk of SARS-CoV-2 infection. However, there are few data on vaccine hesitancy/acceptance and COVID-19 self-protective behaviors among this population. Youth aged 15–24 years (n = 440), predominantly African American and Latine (73%, n = 320) SGM, from Los Angeles and New Orleans reported their vaccine attitudes and COVID-19 and HIV preventive behaviors in October 2020. Latent class analyses categorized individuals into groups based on their vaccine attitudes and preventive behaviors. Relationships between these groups and other factors were analyzed using Fisher’s exact tests, ANOVA, and logistic regression. Most youth had accepting vaccine attitudes (70.2%, n = 309), with 20.7% hesitant (n = 91), and 9.1% resistant (n = 40). SGM and African Americans were significantly less accepting than their cis-gender and heterosexual peers. About two-thirds (63.2%, n = 278) of the respondents reported consistent COVID-19 self-protective behaviors. Youth with pro-vaccine attitudes were most consistently self-protective; however, only 54.4% (n= 168/309) intended to take a COVID-19 vaccine. Homelessness history, race, and sexual orientation were associated with vaccine attitudes. Accepting vaccine attitudes and consistent COVID-19 self-protective behaviors were closely related. COVID-19 attitudes/behaviors were not associated with HIV risk and only loosely associated with SARS-CoV-2 vaccine intentions.

Background: Globally, invasive amphibians are known for their environmental and social impacts that range from poisoning of local fauna and human populations to direct predation on other amphibians. Although several countries on most continents have had multiple introductions of many species, southern Africa appears to have escaped allochthonous introductions. Instead, it has a small number of domestic exotic species that have rapidly expanded their ranges and established invasive populations within South Africa. Objectives & methods: We used the literature to provide a historical overview of dispersal by some of the world’s major invasive amphibians, give examples of species that are commonly moved as stowaways and discuss historical and current amphibian trade in the region. In addition, we give an overview of new South African legislation and how this is applied to amphibian invasions, as well as providing updates on the introduced populations of three domestic exotics: Hyperolius marmoratus, Sclerophrys gutturalis and Xenopus laevis. Results: We show that frogs are mainly moved around southern Africa through ‘jump’ dispersal, although there are a number of records of ‘cultivation’, ‘leading-edge’ and ‘extreme long-distance’ dispersal types. Important pathways include trade in fruit and vegetables, horticultural products and shipping containers. Conclusion: We suggest that southern Africa is becoming more vulnerable to amphibian invasions because of an increase in trade, agricultural and domestic impoundments as well as global climate change. Increasing propagule pressure suggests that preventing new introductions will become a key challenge for the future. Currently, trade in amphibians in the region is practically non-existent, suggesting potential for best practice to prevent importation of species with high invasion potential and to stop the spread of disease.

Os sociólogos do conhecimento tentam explicar as crenças dos agentes epistêmicos por meio da posição social destes no interior de uma hierarquia de relações de poder. O artigo propõe um modo alternativo de conceber a posição social dos agentes epistêmicos. Inspirado nas noções durkheimianas de integração social e de densidade social, o autor define a posição social de um agente com base na densidade de suas relações no interior de redes sociais. Para a sociologia do conhecimento científico em particular, são abordadas as relações sociais no interior de redes profissionais de cientistas. Em seguida, verifica-se a aplicabilidade desse conceito de posição social à sociologia do conhecimento em geral.

In this work the effect of weight reduction using advanced lightweight materials on the mass, energy use, and cost of conventional and battery electric passenger vehicles is compared. Analytic vehicle simulation is coupled with cost assessment to find the optimal degree of weight reduction minimizing manufacturing and total costs. The results show a strong secondary weight and cost saving potential for the battery electric vehicles, but a higher sensitivity of vehicle energy use to mass reduction for the conventional vehicle. Generally, light weighting has the potential to lower vehicle costs, however, the results are very sensitive to parameters affecting lifetime fuel costs for conventional and battery costs for electric vehicles. Based on current technology cost estimates it is shown that the optimal amount of primary mass reduction minimizing total costs is similar for conventional and electric vehicles and ranges from 22% to 39%, depending on vehicle range and overall use patterns. The difference between the optimal solutions minimizing manufacturing versus total costs is higher for conventional than battery electric vehicles.

The objective of this study was to investigate the presence of equine coronavirus (ECoV) in clinical samples submitted to a diagnostic laboratory in Ireland. A total of 424 clinical samples were examined from equids with enteric disease in 24 Irish counties between 2011 and 2015. A real-time reverse transcription polymerase chain reaction was used to detect ECoV RNA. Nucleocapsid, spike and the region from the p4.7 to p12.7 genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. Five samples (1.2%) collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal. Sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p12.7 genes were highly conserved and were closely related to ECoVs identified in other countries. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions. The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland.

This study aims to evaluate the prevalence of mycotoxins in industry-submitted cool-season barley and wheat grown under low heat unit climate conditions. Seventy-two barley samples and 83 wheat samples were submitted by producers and industry from May 2016 to May 2017. The concentrations of twelve common mycotoxins, including nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), ochratoxin A (OTA), zearalenone (ZEN), α-zearalenol (α-ZAL), β-zearalenol (β-ZAL), diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2), and aflatoxin B1 (AFB1), were determined using the liquid chromatography/tandem mass spectrometry method. Mycotoxins were detected in 40 barley (56%) and 35 wheat (42%) samples submitted by producers and industry. DON showed the highest incidence in barley (44%) and wheat (33%). None of the barley samples contained detectable DAS and no wheat samples tested positive for α-ZAL, DAS, T-2, or AFB1. Co-occurrence of DON and other mycotoxins was frequently observed. Among the mycotoxin-positive samples, 70% of barley samples and 54% of wheat samples were co-contaminated with at least two mycotoxins. Four barley (6%) and five wheat (6%) samples contained levels of DON above 1000 μg/kg (regulatory level in diets for lactating dairy animals) and HT-2 content in five barley (7%) and four wheat (5%) samples exceeded 100 μg/kg (regulatory level in diets for cattle and poultry). Overall, contamination of these mycotoxins was more frequent and more severe in barley in comparison with wheat that was submitted by producers and industry. Comprehensive strategies, including the prevention of Fusarium toxins contamination, the routine monitoring of their prevalence, the detoxification of them in feed and food, as well as the inhibition of their absorption in the gastrointestinal tract, are highly required. A rapid detection method needs to be developed to screen mycotoxins in industry-submitted cool-season cereal grains.

Improving powertrain efficiency and reducing vehicle weight are two options for reducing vehicle energy use, emissions, and operating cost that often increase the purchase cost of passenger vehicles. Increasing drivetrain efficiency shrinks the potential for reducing energy use by lightweighting and conversely lighter vehicles benefit less from efficiency improvement. This paper describes a methodology for finding the optimum combination of light-weighting and efficiency measures to achieve minimum lifetime vehicle cost. Using representative technology cost assumptions for a mid-sized passenger car, marginal efficiency improvement in the range of 20 to 30% and lightweighting between 200 and 600 kg are optimal, depending strongly on marginal cost curve characteristics. A reduction in the total cost of vehicle ownership of between 18 and 42% is possible for the representative technology implementation scenarios. In the absence of reliable cost data, a general strategy of designing lightweight vehicles with lower powertrain efficiency or else higher-efficiency powertrains which are integrated in heavier vehicles is recommended.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC ( Pdx1-Cre , LSL-Kras G12D/+ , LSL-Trp53 R172H/+ ) and poorly metastatic KP fl C ( Pdx1-Cre , LSL-Kras G12D/+ , Trp53 fl/+ ) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KP fl C, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.

Although lithium-ion batteries have transformed energy storage, there is a need to develop battery technologies with improved performance. Fluoride-ion batteries (FIBs) may be promising alternatives in part due to their high theoretical energy density and natural elemental abundance. However, electrode materials for FIBs, particularly cathodes, have not been systematically evaluated, limiting rapid progress. Here, we evaluate ternary fluorides from the Materials Project crystal structure database to identify promising cathode materials for FIBs. Structures are further assessed based on stability and whether fluorination/defluorination occurs without unwanted disproportionation reactions. Properties are presented for pairs of fluorinated/defluorinated materials including theoretical energy densities, cost approximations, and bandgaps. We aim to supply a dataset for extracting property and structural trends of ternary fluoride materials that may aid in the discovery of next-generation battery materials., (© 2023. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow–induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival., Competing Interests: Declaration of interests P.T. receives reagents from Kadmon, INXMED (consultant), CRUK Astra Zeneca Alliance laboratory, RedX Pharma, Equlibre Biopharmaceuticals, and Amplia Therapeutics. Under a licensing agreement between Amplia Therapeutics and the Garvan Institute of Medical Research, K.M., D.H., and P.T. (consultant) are entitled to milestone payments. The Owen Sansom laboratory receives funding from Cancer Research Technology, Redex, Astra Zeneca, and Novartis. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

The small GTPase RhoA is a master regulator of signalling in cell-extracellular matrix interactions. RhoA signalling is critical to many cellular processes including migration, mechanotransduction, and is often disrupted in carcinogenesis. Investigating RhoA activity in a native tissue environment is challenging using conventional biochemical methods; we therefore developed a RhoA-FRET biosensor mouse, employing the adaptable nature of intravital imaging to a variety of settings. Mechanotransduction was explored in the context of osteocyte processes embedded in the calvaria responding in a directional manner to compression stress. Further, the migration of neutrophils was examined during in vivo "chemotaxis" in wound response. RhoA activity was tightly regulated during tissue remodelling in mammary gestation, as well as during mammary and pancreatic carcinogenesis. Finally, pharmacological inhibition of RhoA was temporally resolved by the use of optical imaging windows in fully developed pancreatic and mammary tumours in vivo . The RhoA-FRET mouse therefore constitutes a powerful tool to facilitate development of new inhibitors targeting the RhoA signalling axis.

Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene , a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data., Competing Interests: SW, MN, AM, YM, DH, IM, CV, JC, PM, TC, YW, JM, HW, DS, KA, TP, MR, PT No competing interests declared, (© 2018, Warren et al.)

Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the single-cell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin., Competing Interests: PWG is a Director on the Board of Novogen, a company commercialising drugs that are directed against Tpm3.1. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

This paper demonstrates multiphoton excited fluorescence imaging through a polarisation maintaining multicore fiber (PM-MCF) while the fiber is dynamically deformed using all-proximal detection. Single-shot proximal measurement of the relative optical path lengths of all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF. An experimental validation of this approach is presented that compares the proximally measured deformation-induced lateral shift in focal spot position to an independent distally measured ground truth. The proximal measurement of deformation-induced shift in focal spot position is applied to correct for deformation-induced shifts in focal spot position during raster-scanning multiphoton excited fluorescence imaging.

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3'-phosphoinositide accumulation.

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.

The synthesis of ordered mesoporous metal composites and ordered mesoporous metals is a challenge because metals have high surface energies that favor low surface areas. We present results from the self-assembly of block copolymers with ligand-stabilized platinum nanoparticles, leading to lamellar CCM-Pt-4 and inverse hexagonal (CCM-Pt-6) hybrid mesostructures with high nanoparticle loadings. Pyrolysis of the CCM-Pt-6 hybrid produces an ordered mesoporous platinum-carbon nanocomposite with open and large pores (>/=10 nanometers). Removal of the carbon leads to ordered porous platinum mesostructures. The platinum-carbon nanocomposite has very high electrical conductivity (400 siemens per centimeter) for an ordered mesoporous material fabricated from block copolymer self-assembly.

To gain insight into the demographics and experiences of dying patients in a West Virginia acute care hospital, a chart review was conducted of all patients dying over a four-week period in 1997. Most of these patients were usually older with chronic progressive diseases. Half the patients underwent life-sustaining treatment in the last three days of life, and most patients had multiple nonpalliative interventions during this period. Interventions were refused or requested to be stopped by patients or families in over one third of the cases. A poor expected outcome was documented for more than two-thirds of the group. Do-Not-Resuscitate orders were written for most patients often within three days of death. Pain assessment was poorly documented. These findings are consistent with other studies of the hospitalized dying. Reasons for the continuation of non-palliative measures in the terminally ill are explored. Incorporation of palliative care programs into the hospital setting is recommended as a means to improve end-of-life care.

To assess concerns and self-reported competencies in end-of-life (EOL) care, we surveyed a random sample of West Virginia physicians and received 255 responses (33%). Those responding identified three major barriers to good EOL care: patient and family demands for all possible treatments, lack of physician education and inadequate financing. Most identified themselves as less than informed about EOL legislative and regulatory issues and were less than satisfied with their EOL symptom management skills. Most reported that their patients would want hospice care at the end of their lives, but indicated that a major reason for not referring a patient to hospice was patient and family denial of approaching death. Most respondents rated the overall quality of EOL care in West Virginia as fair to poor. We conclude that physician respondents recognize the need to improve their knowledge and skills in EOL care to improve the care of the dying in West Virginia.

Background and Purpose: The objective of this study was to examine the relationships between type of entry-level education and selected student variables., Subjects: Students in their final year of education in entry-level bachelor's and master's degree programs in the United States (N = 766) participated in the study., Methods: Questionnaires were mailed to these students, who represented 22 entry-level physical therapy programs selected at random from the December 1991 issue of Physical Therapy. Two-tailed t tests for independent means and chi-square analyses were performed to determine statistical significance for interval data and categorical data, respectively., Results: Five hundred twelve surveys were returned, for a response rate of 66.8%. Master's degree respondents anticipated greater involvement in research and teaching and felt better prepared to practice across a broad spectrum of clinical practice and to perform activities related to research, teaching, management, and direct access practice. Baccalaureate programs, however, appeared to attract a greater percentage of minority individuals (14.9% versus 5.8%, respectively)., Conclusion and Discussion: These results suggest that differences exist between entry-level bachelor's and master's degree students in physical therapy. Findings of this study may have implications for curriculum planning, recruitment and scholarship efforts, and policy formation in physical therapy education.