Fire suppression makes wildfires more severe and accentuates impacts of climate change and fuel accumulation.

Fire suppression is the primary management response to wildfires in many areas globally. By removing less-extreme wildfires, this approach ensures that remaining wildfires burn under more extreme conditions. Here, we term this the "suppression bias" and use a simulation model to highlight how this bias fundamentally impacts wildfire activity, independent of fuel accumulation and climate change. We illustrate how attempting to suppress all wildfires necessarily means that fires will burn with more severe and less diverse ecological impacts, with burned area increasing at faster rates than expected from fuel accumulation or climate change. Over a human lifespan, the modeled impacts of the suppression bias exceed those from fuel accumulation or climate change alone, suggesting that suppression may exert a significant and underappreciated influence on patterns of fire globally. Managing wildfires to safely burn under low and moderate conditions is thus a critical tool to address the growing wildfire crisis.

Practical Characterization Strategies for Comparison, Qualification, and Selection of Cell Viability Detection Methods for Cellular Therapeutic Product Development and Manufacturing.

Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.

COVID-19's impact on visitation behavior to US national parks from communities of color: evidence from mobile phone data.

The widespread COVID-19 pandemic fundamentally changed many people's ways of life. With the necessity of social distancing and lock downs across the United States, evidence shows more people engage in outdoor activities. With the utilization of location-based service (LBS) data, we seek to explore how visitation patterns to national parks changed among communities of color during the COVID-19 pandemic. Our results show that visitation rates to national parks located closer than 347 km to individuals have increased amidst the pandemic, but the converse was demonstrated amongst parks located further than 347 km from individuals. More importantly, COVID-19 has adversely impacted visitation figures amongst non-white and Native American communities, with visitation volumes declining if these communities are situated further from national parks. Our results show disproportionately low-representations amongst national park visitors from these communities of color. African American communities display a particularly concerning trend whereby their visitation to national parks is substantially lower amongst communities closer to national parks.

Exclusionary Effects of Campsite Allocation through Reservations in U.S. National Parks: Evidence from Mobile Device Location Data.

Campsites represent highly sought after recreational amenities in the national parks of the United States. Equitable allocation of scarce recreational resources has long been a key management issue in U.S. national parks, but has become increasingly difficult in an era of increasing demand. At present, a growing number of national park campsites are allocated through an online reservation system well in advance of a camper's arrival at a park. Compounding the challenge of allocating these campsites is a long history of exclusivity within national park camping-institutionalized through campground design and predicated on a legacy of the leisure class's affinity for camping in national parks. Given national park camping's history of exclusivity, this exploratory study seeks to explore how online reservation systems may impact the demographics of national park campers. Using mobile device location data, estimated demographics were calculated for campers in five national park campgrounds in the U.S. that each contained some sites requiring reservations and some sites available on a first-come, first-served basis. We detail results from analyses of variance between campsites requiring reservations and those that are available on a first-come, first-served basis. Results suggest that for each of the five campgrounds, those campers camping in sites that require reservations came from areas with higher median household incomes, on average. In three of the five campgrounds, this difference was significant. Additionally, in an urban-proximate setting, those camping in sites requiring reservations came from areas with a higher portion of White residency than those campers in campsites not requiring reservations, on average. We conclude with discussion that includes management implications concerning the growing prominence of online reservation systems for outdoor recreation amenities, and a brief research agenda for diversity, equity, and inclusion (DEI) as they relate to campgrounds. Principally, the former group of implications includes the realization that online reservation systems present the unintended consequence of excluding low-income, and perhaps non-White, would-be campers-a conclusion drawn from the results of this exploratory study. This discussion includes an analysis of the distributive justice of online reservation systems.

Rapid cell counting and viability detection method of Escherichia coli Nissle using image cytometry.

The improvement of cell enumeration methods for the counting of Escherichia coli (E. coli) is important as E. coli gains in popularity as a basis for biopharmaceutical applications. In the biopharmaceutical industry, enumerating, characterizing, and dosing the accurate number of cells is imperative. In this work, we demonstrate the utilization of a chip-based image cytometer using a thin-gap, low volume counting chamber consumable to directly enumerate E. coli in bright field and fluorescence, and measure their viability using SYTOX™ Green. The total E. coli particles can be counted accurately label-free by adjusting the focus and targeting the linear range of the instrument. The E. coli are stained with SYTOX™ Green to count the membrane-compromised dead bacterial cells in the green fluorescence channel, while the total cells are counted using the bright field channel. Optimization of the system settings, image focus, cell counting range, and staining conditions have yielded a precise, rapid, and accurate E. coli cell enumeration and viability measurement.

Understanding changes in park visitation during the COVID-19 pandemic: A spatial application of big data.

In the spring of 2020, the COVID-19 pandemic changed the daily lives of people around the world. In an effort to quantify these changes, Google released an open-source dataset pertaining to regional mobility trends-including park visitation trends. Changes in park visitation are calculated from an earlier baseline period for measurement. Park visitation is robustly linked to positive wellbeing indicators across the lifespan, and has been shown to support wellbeing during the COVID-19 pandemic. Therefore, this dataset offers vast application potential, containing aggregated information from location data collected via smartphones worldwide. However, empirical analysis of these data is limited. Namely, the factors influencing reported changes in mobility and the degree to which these changes can be directly attributable to COVID-19 remain unknown. This study aims to address these gaps in our understanding of the changes in park visitation, the causes of these changes (e.g., safer-at-home orders, amount of COVID-19 cases per county, climate, etc.) and possible impacts to wellbeing by constructing and testing a spatial regression model. Results suggest that elevation and latitude serve as primary influences of reported changes in park visitation from the baseline period. Therefore, it is surmised that Google's reported changes in park-related mobility are only partially the function of COVID-19.

Big data spatial analysis of campers' landscape preferences: Examining demand for amenities.

Outdoor recreation decision-making has received significant research interest over the last fifty years. In the context of campsite choice, this previous research has almost exclusively used stated preference data and aspatial methods to understand decision-making. This present research seeks to understand how recreationists reach decisions on the selection of campsites and what aspects of the recreational setting drive demand through an examination of a big dataset of revealed preference data using a spatial regression. Specifically, we examine which managerial, social, and ecological aspects of the setting influence demand for campsites in Zion National Park's (USA) Watchman Campground using reservation data from the Recreation Information Database (RIDB). Results indicate that price, access to electricity, ease of access, and proximity to the Virgin River are significantly predictive of demand. Study implications for park management, including campsite allocation and distributive justice, are provided. Additionally, implications for future research methodology, including the use of transaction-style big data in protected area management research, are discussed.

Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells.

Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of the dim and diffuse objects after trypan blue staining. Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrate the co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. The videos clearly show immediate cell rupturing after trypan blue contact. The formation of these diffuse objects was monitored and counted over time as cells die outside of the incubator. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect the outcomes of cellular therapies, which require accurate measurements of immune cells that will be infused back into patients.

Tomographic fluorescence lifetime multiplexing in the spatial frequency domain.

The ability to simultaneously recover multiple fluorophores within biological tissue (multiplexing) can have important applications for tracking parallel disease processes in vivo. Here we present a novel method for rapid and quantitative multiplexing within a scattering medium, such as biological tissue, based on fluorescence lifetime contrast. This method employs a tomographic inversion of the asymptotic (late) portion of time-resolved spatial frequency (SF) domain measurements. Using Monte Carlo simulations and phantom experiments, we show that the SF-asymptotic time domain (SF-ATD) approach provides a several-fold improvement in relative quantitation and localization accuracy over conventional SF-time domain inversion. We also show that the SF-ATD approach can exploit selective filtering of high spatial frequencies to dramatically improve reconstruction accuracy for fluorophores with subnanosecond lifetimes, which is typical of most near-infrared fluorophores. These results suggest that the SF-ATD approach will serve as a powerful new tool for whole-body lifetime multiplexing.

Substrate-based near-infrared imaging sensors enable fluorescence lifetime contrast via built-in dynamic fluorescence quenching elements.

Enzymatic activity sensing in fluorescence lifetime (FLT) mode with "self-quenched" macromolecular near-infrared (NIR) sensors is a highly promising strategy for in vivo imaging of proteolysis. However, the mechanisms of FLT changes in such substrate-based NIR sensors have not yet been studied. We synthesized two types of sensors by linking the near-infrared fluorophore IRDye 800CW to macromolecular graft copolymers of methoxy polyethylene glycol and polylysine (MPEG-gPLL) with varying degrees of MPEGylation and studied their fragmentation induced by trypsin, elastase, plasmin and cathepsins (B,S,L,K). We determined that the efficiency of such NIR sensors in FLT mode depends on sensor composition. While MPEG-gPLL with a high degree of MPEGylation showed rapid (τ1/2=0.1-0.2 min) FLT increase (Δτ=0.25 ns) upon model proteinase-mediated hydrolysis in vivo, lower MPEGylation density resulted in no such FLT increase. Temperature-dependence of fluorescence de-quenching of NIR sensors pointed to a mixed dynamic/static-quenching mode of MPEG-gPLL-linked fluorophores. We further demonstrated that although the bulk of sensor-linked fluorophores were de-quenched due to the elimination of static quenching, proteolysis-mediated deletion of a fraction of short (8-10kD) negatively charged fragments of highly MPEGylated NIR sensor is the most likely event leading to a rapid FLT increase phenomenon in quenched NIR sensors. Therefore, the optimization of "built-in" dynamic quenching elements of macromolecular NIR sensors is a potential avenue for improving their response in FLT mode.

Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.

In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-ß-cyclodextrin (mßCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

In vivo tomographic imaging of deep-seated cancer using fluorescence lifetime contrast.

Preclinical cancer research would benefit from noninvasive imaging methods that allow tracking and visualization of early-stage metastasis in vivo. Although fluorescent proteins revolutionized intravital microscopy, two major challenges that still remain are tissue autofluorescence and hemoglobin absorption, which act to limit intravital optical techniques to large or subcutaneous tumors. Here, we use time-domain (TD) technology for the effective separation of tissue autofluorescence from extrinsic fluorophores, based on their distinct fluorescence lifetimes. In addition, we use cancer cells labeled with near infrared fluorescent proteins (iRFP) to allow deep-tissue imaging. Our results demonstrate that TD imaging allows the detection of metastasis in deep-seated organs of living mice with a more than 20-fold increase in sensitivity compared with conventional continuous wave techniques. Furthermore, the distinct fluorescence lifetimes of iRFPs enable lifetime multiplexing of three different tumors, each expressing unique iRFP labels in the same animal. Fluorescence tomographic reconstructions reveal three-dimensional distributions of iRFP720-expressing cancer cells in lungs and brain of live mice, allowing ready longitudinal monitoring of cancer cell fate with greater sensitivity than otherwise currently possible.

Preclinical whole body time domain fluorescence lifetime multiplexing of fluorescent proteins.

The application of time domain (TD) fluorescence lifetime multiplexing for the detection of fluorescent proteins (FPs) in whole animals, in the presence of a strong background tissue autofluorescence and excitation light leakage is discussed. Tissue autofluorescence (AF) exhibits a nonexponential temporal response, distinct from the mono-exponential decay of FPs. This allows a direct separation of FP fluorescence from AF using a dual basis function approach. We establish the detection limits of this approach using in vitro and in vivo measurements. We also demonstrate, using an experimental model of lymph node metastasis, that FP-AF lifetime multiplexing provides a greater than 30-fold improvement in contrast-to-background ratio compared with continuous wave data. In addition, we show that TD detection can simultaneously discriminate between up to three red shifted FPs placed under the skin of a nude mouse based on their distinct fluorescence lifetimes.

Tomographic lifetime imaging using combined early- and late-arriving photons.

We present a novel, hybrid approach for time domain fluorescence tomography that efficiently combines lifetime multiplexing using late-arriving or asymptotic photons, with the high spatial resolution capability of early photon tomography. We also show that a decay amplitude-based asymptotic approach is superior to direct inversion of late-arriving photons for tomographic lifetime imaging within turbid media. The hybrid reconstruction approach is experimentally shown to recover fluorescent inclusions separated as close as 1.4 mm, with improved resolution and reduced cross talk compared to just using early photons or the asymptotic approach alone.

Resolution below the point spread function for diffuse optical imaging using fluorescence lifetime multiplexing.

We show that asymptotic lifetime-based fluorescence tomography can localize multiple-lifetime targets separated well below the diffuse point spread function of a turbid medium. This is made possible due to a complete diagonalization of the time domain forward problem in the asymptotic limit. We also show that continuous wave or direct time gate approaches to fluorescence tomography are unable to achieve this separation, indicating the unique advantage of a decay-amplitude-based approach for tomographic lifetime multiplexing with time domain data.

High resolution helium ion scanning microscopy of the rat kidney.

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.

The PEG-fluorochrome shielding approach for targeted probe design.

We provide a new approach for fluorescent probe design termed "PEG-fluorochrome shielding", where PEGylation enhances quantum yields while blocking troublesome interactions between fluorochromes and biomolecules. To demonstrate PEG-fluorochrome shielding, fluorochrome-bearing peptide probes were synthesized, three without PEG and three with a 5 kDa PEG functional group. In vitro, PEG blocked the interactions of fluorochrome-labeled peptide probes with each other (absorption spectra, self-quenching) and reduced nonspecific interactions with cells (by FACS). In vivo, PEG blocked interactions with biomolecules that lead to probe retention (by surface fluorescence). Integrin targeting in vivo was obtained as the differential uptake of an (111)In-labeled, fluorochrome-shielded, integrin-binding RGD probe and a control RAD. Using PEG to block fluorochrome-mediated interactions, rather than synthesizing de novo fluorochromes, can yield new approaches for the design of actively or passively targeted near-infrared fluorescent probes.

Differential, phosphorylation dependent trafficking of AQP2 in LLC-PK1 cells.

The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture.

Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields.

BACKGROUND: Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood. METHODS: The purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs) undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF) and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR) were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP) and collagen type 1 (col1), and stress response markers, such as heat shock protein 27 (hsp27) and heat shock protein 70 (hsp70). Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG) imaging. RESULTS: Quantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p < 0.05) in samples exposed to the electric field. A delayed but two fold increase in ALP and col1 transcript was detected by week 2 (p < 0.05) in differentiating hMSCs exposed to an electric field in comparison to the nonstimulated controls. Upregulation in stress marker, hsp27, and type 1 collagen deposition were correlated with this response. Increases in NADH, FAD, and lipofuscin were traced in the stimulation group during the first week of field exposure with differences statistically significant on day 10 (p < 0.05). Changes in hsp27 expression correlate well with changes in lipofuscin detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28. CONCLUSIONS: Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.

Development of an in vitro model to study the impact of BMP-2 on metastasis to bone.

Prostate cancer cases and deaths have increased for years, yet the mechanisms involved in prostate cancer metastasis to bone remain poorly understood. To address this need, an effective and relevant in vitro model for the study of prostate cancer bone metastases would be useful. Therefore, a 3D in vitro tissue system was established using prostate cancer cells (PC3), suitable culture conditions and a 3D silk scaffold biomaterial to provide mechanically robust and slow degrading matrices to support the tissues for extended time frames. The role of BMP-2 on the progression of prostate cancer was investigated using this 3D tissue system. The results suggest that BMP-2 stimulates the migration of PC3 cells, suggesting insight into mechanisms involved in this critical step in the disease. The data support the conclusion that this in vitro system mimics aspects of prostate cancer metastasis in the presence of BMP-2, thus the system can be utilized as a starting point as an in vitro model for further studies of prostate cancer development and metastasis, as well as in the screening of new therapeutic treatments.