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Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers.

Bortezomib (VELCADE) could sensitize certain human renal cell carcinoma (RCC) lines to the apoptotic effects of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Analysis of seven human RCC showed a clear increase in the sensitivity of four of the RCC to TRAIL cytotoxicity following bortezomib (5-20 nmol/L) treatment, whereas the remaining three remained resistant. Tumor cell death following sensitization had all the features of apoptosis. The enhanced antitumor activity of the bortezomib and TRAIL combination was confirmed in long-term (6 days) cancer cell outgrowth assays. The extent of proteasome inhibition by bortezomib in the various RCC was equivalent. Following bortezomib treatment, neither changes in the intracellular protein levels of various Bcl-2 and IAP family members, nor minor changes in expression of TRAIL receptors (DR4, DR5), correlated well with the sensitization or resistance of RCC to TRAIL-mediated apoptosis. However, enhanced procaspase-8 activation following bortezomib pretreatment and subsequent TRAIL exposure was only observed in the sensitized RCC in both cell extracts and death-inducing signaling complex immunoprecipitates. These data suggest that the molecular basis for bortezomib sensitization of RCC to TRAIL primarily involves early amplification of caspase-8 activity. In the absence of this increased caspase-8 activation, other bortezomib-induced changes are not sufficient to sensitize RCC to TRAIL-mediated apoptosis. Mol Cancer Res; 8(5); 729–38. ©2010 AACR.

Supplementary Data from Bortezomib Sensitizes Human Renal Cell Carcinomas to TRAIL Apoptosis through Increased Activation of Caspase-8 in the Death-Inducing Signaling Complex

Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand–dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8–dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome.Significance:These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies.

Supplementary Materials & Methods, Supplementary Figures 1-10 with Figure Legends

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c+CD8+ dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8+ T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2−/− mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy. Cancer Res; 75(24); 5260–72. ©2015 AACR.

Supplementary Figure 5 from Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity

Supplementary Figure 3 from Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity

Supplementary Figure 2 from Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity

Supplementary Figure 6 from Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity

Calreticulin expression on bortezomib-treated tumor cells.

Legends for Supplementary Figures S1-S3.

Supplementary Figure 1 from Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity

Supplementary Figure 4 from Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity

Physachenolide C (1) is a 17β-hydroxywithanolide natural product with a unique anticancer potential, as it exhibits potent and selective in vitro antiproliferative activity against prostate cancer (PC) cells and promotes TRAIL-induced apoptosis of renal carcinoma (RC) and poly I:C-induced apoptosis of melanoma cells. To explore the effect of ring A/B modifications of physachenolide C (1) on these biological activities, 23 of its natural and semisynthetic analogues were evaluated. Analogues 4-23 were prepared by chemical transformations of a readily accessible compound, physachenolide D (2). Compound 1 and its analogues 2-23 were evaluated for their antiproliferative activity against PC (LNCaP and 22Rv1), RC (ACHN), and melanoma (M14 and SK-MEL-28) cell lines and normal human foreskin fibroblast (HFF) cells. Most of the active analogues had selective and potent activity in reducing cell number for PC cell lines, some showing selectivity for androgen-independent and enzalutamide-resistant 22Rv1 cells compared to androgen-dependent LNCaP cells. Analogues with IC50s below 5.0 μM against ACHN cells, when tested in the presence of TRAIL, showed a significantly increased ability to reduce cell number, and those analogues active against the M14 and SK-MEL-28 cell lines exhibited enhanced activity when combined with poly I:C. These data provide additional structure-activity relationship information for 17β-hydroxywithanolides and suggest that selective activities of some analogues may be exploited to develop natural products-based tumor-specific agents for cancer chemotherapy.

Saltmarsh sparrows ( Ammospiza caudacuta ) and seaside sparrows ( A. maritima ) are species of conservation concern primarily due to global sea-level rise and habitat degradation. Environmental mercury (Hg) contamination may present additional threats to their reproductive success and survival. To assess site-specific total mercury (THg) exposure and identify environmental correlates of THg detection across a large portion of the breeding range where these species co-occur, we sampled blood from adult male saltmarsh and seaside sparrows at 27 marsh sites from Maine to Virginia, USA. The mean THg concentration (±1 SD) throughout the entire sampling range was 0.531 ± 0.287 µg/g wet weight (ww) for saltmarsh sparrows and 0.442 ± 0.316 µg/g ww for seaside sparrows. Individual THg concentrations ranged from 0.135–1.420 µg/g ww for saltmarsh sparrows and 0.153–1.530 µg/g ww for seaside sparrows. Model averaging from a suite of linear mixed models supported species-based differences in blood THg. On average, saltmarsh sparrows had 20.1% higher blood THg concentrations than seaside sparrows. We hypothesize that species-specific THg concentrations are influenced by differences in diet or foraging preferences between these species. We did not detect any correlations between sparrow THg concentrations and land cover characteristics surrounding sampled marshes or cumulative average annual precipitation.

Background Flowers which imitate insect oviposition sites probably represent the most widespread form of floral mimicry, exhibit the most diverse floral signals and are visited by two of the most speciose and advanced taxa of insect – beetles and flies. Detailed comparative studies on brood-site mimics pollinated exclusively by each of these insect orders are lacking, limiting our understanding of floral trait adaptation to different pollinator groups in these deceptive systems. Methods Two closely related and apparent brood-site mimics, Typhonium angustilobum and T. wilbertii (Araceae) observed to trap these distinct beetle and fly pollinator groups were used to investigate potential divergence in floral signals and traits most likely to occur under pollinator-mediated selection. Trapped pollinators were identified and their relative abundances enumerated, and thermogenic, visual and chemical signals and morphological traits were examined using thermocouples and quantitative reverse transcription–PCR, reflectance, gas chromatography–mass spectrometry, floral measurements and microscopy. Key Results Typhonium angustilobum and T. wilbertii were functionally specialized to trap saprophagous Coleoptera and Diptera, respectively. Both species shared similar colour and thermogenic traits, and contained two highly homologous AOX genes (AOX1a and AOX1b) most expressed in the thermogenic tissue and stage (unlike pUCP). Scent during the pistillate stage differed markedly – T. angustilobum emitted a complex blend of sesquiterpenes, and T. wilbertii, a dung mimic, emitted high relative amounts of skatole, p-cresol and irregular terpenes. The species differed significantly in floral morphology related to trapping mechanisms. Conclusions Functional specialization and pollinator divergence were not associated with differences in anthesis rhythm and floral thermogenic or visual signals between species, but with significant differences in floral scent and morphological features, suggesting that these floral traits are critical for the attraction and filtering of beetle or fly pollinators in these two brood-site mimics.

Context. Pressure profiles are sensitive probes of the thermodynamic conditions and the internal structure of galaxy clusters. The intra-cluster gas resides in hydrostatic equilibrium within the dark-matter gravitational potential. However, this equilibrium may be perturbed; for example, as a consequence of thermal energy losses, feedback, and non-thermal pressure supports. Accurate measures of the gas pressure over cosmic time are crucial for constraining cluster evolution as well as the contributions from astrophysical processes. Aims. In this work we present a novel algorithm for deriving the pressure profiles of galaxy clusters from the Sunyaev-Zeldovich (SZ) signal measured on a combination of Planck and South Pole Telescope (SPT) observations. The synergy of the two instruments makes it possible to track the profiles on a wide range of spatial scales. We exploited the sensitivity of the Planck High-Frequency Instrument to the larger scales in order to observe the faint peripheries, and took advantage of the higher spatial resolution of SPT to solve the innermost regions. Methods. We developed a two-step pipeline to take advantage of the specifications of each instrument. We first performed a component separation on the two data sets separately in order to remove the background (CMB) and foreground (Galactic emission) contaminants. We then jointly fitted a parametric pressure profile model on a combination of Planck and SPT data. Results. We validated our technique on a sample of six CHEX-MATE clusters detected by SPT. We compare the results of the SZ analysis with profiles derived from X-ray observations with XMM-Newton. We find excellent agreement between these two independent probes of the gas pressure structure.

Freshwater phytoplankton carbon fixation is an important water quality parameter that can provide information about the health of lake ecosystems as well as their impacts on regional carbon cycling...

The excitation and detection of coherent phonons has given unique insights into condensed matter, in particular for materials with strong electron-phonon coupling. We report a study of coherent phonons in the layered charge density wave (CDW) compound 1$T$-TaSe$_2$ performed using transient broadband reflectivity spectroscopy, in the photon energy range 1.75-2.65 eV. Several intense and long lasting (> 20 ps) oscillations, arising from the CDW superlattice reconstruction, are observed allowing for detailed analysis of the spectral dependence of their amplitude and phase. We find that for energies above 2.4 eV, where transitions involve Ta d-bands, the CDW amplitude mode at 2.19 THz dominates the coherent response. At lower energies, instead, beating arises between additional frequencies, with a particularly intense mode at 2.95 THz. Interestingly, our spectral analysis reveals a $π$ phase shift at 2.4 eV. Results are discussed considering the selective coupling of specific modes to energy bands involved in the optical transitions seen in steady-state reflectivity. The work demonstrates how coherent phonon spectroscopy can distinguish and resolve optical states strongly coupled to the CDW order and provide additional information normally hidden in conventional steady-state experiments., 20 pages, 4 figures

With the aim of designing new metallosupramolecular architectures for drug delivery, research has focused on porous 3-dimensional (3D)-metallacages able to encapsulate cytotoxic agents protecting them from metabolism while targeting them to cancer sites. Here, two self-assembled [Pd2L4]4+ cages (CG1 and CG2) featuring 3,5-bis(3-ethynylpyridine)phenyl ligands (L) exo-functionalised with dipyrromethene (BODIPY) groups have been synthesised and characterised by different methods, including NMR spectroscopy and mass spectrometry. 1H NMR spectroscopy studies shows that the cages are able to encapsulate the anticancer drug cisplatin in their hydrophobic cavity, as evidenced by electrostatic potential (ESP) analysis based on XRD studies. The stability of the cages in an aqueous environment, and in the presence of the intracellular reducing agent glutathione, has been confirmed by UV-visible absorption spectroscopy. The luminescence properties of the cages enabled the investigation of their cellular uptake and intracellular localisation in human cancer cells by confocal laser scanning microscopy. In melanoma A375 cells, cage CG1 is taken up via active transport and endocytic trafficking studies show little evidence of transport through the early endosome while the cages accumulated in melanosomes rather than lysosomes. The antiproliferative activity of the lead cage was investigated in A375 together with two breast cancer cell lines, SK-BR-3 and MCF7. While the cage per se is non-cytotoxic, very different antiproliferative effects with respect to free cisplatin were evidenced for the [(cisplatin)2⊂CG1·BF4] complex in the various cell lines, which correlate with its different intracellular localisation profiles. The obtained preliminary results provide a new hypothesis on how the subcellular localisation of the cage affects the cisplatin intracellular release.

© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society. The process of pollinator-driven evolution is best studied at the level of populations and among closely related plant species. Araceae provide a plant model for investigating plant-pollinator interactions, but few studies have investigated intraspecific variation in their pollination systems. Typhonium brownii (Araceae) is distributed widely across 2000 km from tropical to temperate latitudes in Australia, yet the existence of intraspecific variation and ecotypes has not been investigated. Typhonium brownii from five regions, potentially representing distinct taxa, and populations of the sister species, T. eliosurum, were studied to explore pollinator and floral trait divergence. We characterize significant intraspecific floral trait variation in T. brownii, indicating the existence of a species complex, despite the taxa trapping similar Coleoptera (Staphylinidae, Scarabaeidae). Although all T. brownii showed similar temperature increases in the appendix, there were significant shifts in the timing and pattern of thermogenic and anthesis rhythms between regions (taxa), and all T. brownii taxa had distinct scent compositions, with T. sp. aff. brownii being the most dissimilar to other taxa. In contrast, T. eliosurum inflorescences almost exclusively trapped Diptera (Sphaeroceridae, Psychodidae), had modest temperature increases confined to the staminate zone and had a distinct scent profile which differed from all T. brownii taxa; this scent was confirmed in field bioassays to be important for pollinator attraction. Prevalent volatile organic compounds (VOCs) emitted by T. eliosurum and T. brownii taxa included the common dung constituents skatole, indole and p-cresol. Typhonium eliosurum and T. brownii taxa further differed significantly in morphology and trapping mechanisms, particularly the fly-pollinated T. eliosurum. It is possible that a subset of ubiquitous VOCs identified in T. eliosurum and T. brownii taxa attract local communities of dung-seeking flies and beetles, and that floral morphological features are more important for trapping different insect orders in these dung mimics.

Membrane-active peptides have been extensively studied to probe protein–membrane interactions, to act as antimicrobial agents and cell-penetrating peptides (CPPs) for the delivery of therapeutic agents to cells. Hundreds of membrane-active sequences acting as CPPs have now been described including bioportides that serve as single entity modifiers of cell physiology at the intracellular level. Translation of promising CPPs in pre-clinical studies have, however, been disappointing as only few identified delivery systems have progressed to clinical trials. To search for novel membrane-active peptides a sequence from the EGFR juxtamembrane region was identified (named EJP18), synthesised, and examined in its L- and D-form for its ability to mediate the delivery of a small fluorophore and whole proteins to cancer cell lines. Initial studies identified the peptide as being highly membrane-active causing extensive and rapid plasma membrane reorganisation, blebbing, and toxicity. At lower, non-toxic concentrations the peptides outperformed the well-characterised CPP octaarginine in cellular delivery capacity for a fluorophore or proteins that were associated with the peptide covalently or via ionic interactions. EJP18 thus represents a novel membrane-active peptide that may be used as a naturally derived model for biophysical protein–membrane interactions or for delivery of cargo into cells for therapeutic or diagnostic applications.

Efficacious anticancer therapies for targeting plasma membrane receptors with antibody based therapeutics are often contingent on sufficient endocytic delivery of receptor and conjugate to lysosomes. This results in downregulation of receptor activity and, in the case of antibody-drug conjugates (ADCs), intracellular release of a drug payload. The oncogenic receptor HER2 is a priority therapeutic target in breast cancer. Known as an “endocytosis resistant” receptor, HER2 thwarts the receptor downregulating efficiency of the frontline treatment trastuzumab and reduces the potential of trastuzumab-based therapies such as trastuzumab-emtansine. We previously demonstrated that strategically inducing trastuzumab and HER2 crosslinking in breast cancer cells promoted endocytosis and lysosomal delivery of the HER2-trastuzumab complex, stimulating downregulation of the receptor. Here we reveal that HER3, but not EGFR, is also concomitantly downregulated with HER2 after crosslinking. This is accompanied by strong activation of MEK/ERK pathway that we show does not directly contribute to HER2/trastuzumab endocytosis. We show that crosslinking induced trastuzumab endocytosis occurs via clathrin-dependent and independent pathways and is an actin-dependent process. Detailed ultrastructural studies of the plasma membrane highlight crosslinking-specific remodelling of microvilli and induction of extensive ruffling. Investigations in a cell model of acquired trastuzumab resistance demonstrate, for the first time, that they are refractory to crosslinking induced HER2 endocytosis and downregulation. This implicates further arrest of HER2 internalisation in developing trastuzumab resistance. Overall our findings highlight the potential of receptor crosslinking as a therapeutic strategy for cancer while exposing the ability of cancer cells to develop resistance via endocytic mechanisms.

Gold nanoparticles have been researched for many biomedical applications in diagnostics, theranostics, and as drug delivery systems. When conjugated to fluorophores, their interaction with biological cells can be studied in situ and real time using fluorescence microscopy. However, an important question that has remained elusive to answer is whether the fluorophore is a faithful reporter of the nanoparticle location. Here, our recently developed four-wave-mixing optical microscopy is applied to image individual gold nanoparticles and in turn investigate their co-localisation with fluorophores inside cells. Nanoparticles from 10 nm to 40 nm diameter were conjugated to fluorescently-labeled transferrin, for internalisation via clathrin-mediated endocytosis, or to non-targeting fluorescently-labelled antibodies. Human (HeLa) and murine (3T3-L1) cells were imaged at different time points after incubation with these conjugates. Our technique identified that, in most cases, fluorescence originated from unbound fluorophores rather than from fluorophores attached to nanoparticles. Fluorescence detection was also severely limited by photobleaching, quenching and autofluorescence background. Notably, correlative extinction/fluorescence microscopy of individual particles on a glass surface indicated that commercial constructs contain large amounts of unbound fluorophores. These findings highlight the potential problems of data interpretation when reliance is solely placed on the detection of fluorescence within the cell, and are of significant importance in the context of correlative light electron microscopy.

Cyclization of cell-penetrating peptides (CPPs) often results in improved capacity for intracellular delivery of a range of cargoes but quantitating the distinct subcellular localization of them, and their linear counterparts, remains a challenge. Here we describe an optimized method for recombinant generation and purification of eGFP attached to the cyclic form of the newly discovered CPP EJP18 in E. coli. We also demonstrate a novel microscopy method for quantifying its subcellular distribution in leukemia cells.

Cyclization of cell-penetrating peptides (CPPs) often results in improved capacity for intracellular delivery of a range of cargoes but quantitating the distinct subcellular localization of them, and their linear counterparts, remains a challenge. Here we describe an optimized method for recombinant generation and purification of eGFP attached to the cyclic form of the newly discovered CPP EJP18 in E. coli. We also demonstrate a novel microscopy method for quantifying its subcellular distribution in leukemia cells.

We present a detailed study of the bulk electronic structure of high-quality VSe2 single crystals using optical spectroscopy. Upon entering the charge density wave phase below the critical temperature of 112 K, the optical conductivity of VSe2 undergoes a significant rearrangement. A Drude response present above the critical temperature is suppressed while a new interband transition appears around 0.07 eV. From our analysis, we estimate that part of the spectral weight of the Drude response is transferred to a collective mode of the CDW phase. The remaining normal-state charge dynamics appears to become strongly damped by interactions with the lattice as evidenced by a mass enhancement factor m∗/ m ≈ 3. In addition to the changes taking place in the electronic structure, we observe the emergence of infrared active phonons below the critical temperature associated with the 4a × 4a lattice reconstruction.

O_LIBirds are frequently used as indicator taxon in ecology, but traditional surveys are prone to error and largely inadequate in the dense, speciose tropical forests where they are most needed. Passive acoustic monitoring (PAM) has the potential to address many of these pitfalls, providing unique advantages for both long-term monitoring and rapid biodiversity assessment. Here, we evaluate the effectiveness of PAM for characterizing spatiotemporal variation in avian species richness, diversity, vocal activity, and community composition in the Amazon basin global biodiversity hotspot. C_LIO_LIWe employed an array of ten autonomous recording units (ARUs) spanning edge, degraded, and interior forest at a reserve on the deforestation frontier of Madre de Dios, Peru, a region featuring some of the richest, most complex avian assemblages on Earth. Recordings from 21 dawn-hours across three days at seven sites were manually annotated by a single observer. C_LIO_LIARUs and manual annotation performed well as an inventory method, especially for midstory and understory species. Sites in edge and degraded habitats featured significantly lower avian species richness, Shannon diversity, and vocal activity levels than in the forest interior, patterns that replicate the findings of prior high-effort avian censuses in the region. We observed significant temporal variation between days; at all featured sites, vocal activity was highest on January 20th and lowest on January 31st. C_LIO_LIWe demonstrated that novel annotation-generated metrics can work as effective proxies for abundance data and per-capita song rate, correlating with diversity indices and efficiently characterizing habitats at a level of detail unobtainable with human observers. Generalist species were significantly overrepresented in the soundscapes of interior forest, relative to their contributed share of species richness, and interior specialists were underrepresented, suggesting that the entire 191ha site is degraded. The between-days temporal variation that we documented, almost certainly overlooked without simultaneous monitoring, may obfuscate or distort the results of traditional surveys. C_LIO_LISynthesis & Applications. We propose that PAM should become a cornerstone of biodiversity research. The standardization advantages demonstrated in this study, permanent storage, multi-taxa applications, and potential of automated identification make bioacoustics an ideal methodological avenue for guiding management and policy under rapid global change. C_LI

Saltmarsh sparrows (Ammospiza caudacuta) and seaside sparrows (A. maritima) are species of conservation concern primarily due to global sea-level rise and habitat degradation. Environmental mercury (Hg) contamination may present additional threats to their reproductive success and survival. To assess site-specific total mercury (THg) exposure and identify environmental correlates of THg detection, we sampled blood from adult male saltmarsh and seaside sparrows at 27 sites between Maine and Virginia, USA. The mean THg concentration (±1 SD) throughout the entire sampling range was 0.531 ± 0.287 µg/g wet weight (ww) for saltmarsh sparrows and 0.442 ± 0.316 µg/g ww for seaside sparrows. Individual THg concentrations ranged from 0.135-1.420 µg/g ww for saltmarsh sparrows and 0.153-1.530 µg/g ww for seaside sparrows. Model averaging from a suite of linear mixed models showed that saltmarsh sparrows averaged 20.1% higher blood THg concentrations than seaside sparrows, potentially due to differences in diet or foraging behavior. We found no evidence for a relationship between sparrow THg concentrations and land cover surrounding sampled marshes or average precipitation-based Hg deposition. Overall, our results suggest considerable, unexplained variation in tidal marsh sparrow blood THg concentrations over their co-occurring breeding ranges.

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

Lake Erie has experienced dramatic changes in water quality over the past several decades requiring extensive monitoring to assess effectiveness of adaptive management strategies. Remote sensing offers a unique potential to provide synoptic monitoring at daily time scales complementing in-situ sampling activities occurring in Lake Erie. Bio-optical remote sensing algorithms require knowledge about the inherent optical properties (IOPs) of the water for parameterization to produce robust water quality products. This study reports new IOP and apparent optical property (AOP) datasets for western Lake Erie that encapsulate the May–October period for 2015 and 2016 at weekly sampling intervals. Previously reported IOP and AOP observations have been temporally limited and have not assessed statistical differences between IOPs over spatial and temporal gradients. The objective of this study is to assess trends in IOPs over variable spatial and temporal scales. Large spatio-temporal variability in IOPs was observed between 2015 and 2016 likely due to the difference in the extent and duration of mid-summer cyanobacteria blooms. Differences in the seasonal trends of the specific phytoplankton absorption coefficient between 2015 and 2016 suggest differing algal assemblages between the years. Other IOP variables, including chromophoric, dissolved organic matter (CDOM) and beam attenuation spectral slopes, suggest variability is influenced by river discharge and sediment re-suspension. The datasets presented in this study show how these IOPs and AOPs change over a season and between years, and are useful in advancing the applicability and robustness of remote sensing methods to retrieve water quality information in western Lake Erie.

Water quality issues continue to impact recreational and resource use of waterways in the Great Lakes and across smaller inland water bodies in their watersheds. With the advancements in small unmanned aerial systems (sUAS) and in small sensor production, sUAS offer flexibility to overcome several of the shortcomings of satellite and airborne systems, and complement their measurements in this environment. In this study, we deployed two different low-cost, boat launchable sUAS configurations instrumented with Ocean Optics STS hyperspectral Vis-NIR spectroradiometers capable of making measurements over an approximately 2.5 km2 area, below the cloud deck in the nearshore and open lake environment. Flights of these systems were conducted at ten locations over Lake Erie and the Maumee River. Measured spectra compared well with at-surface based Analytical Spectral Devices (ASD) Fieldspec measured spectra, and derived parameters were consistent with in-water FluoroProbe measured water quality parameters and field observations. Using flight data, we constructed transect maps of derived CI products which show the variability in algae abundance in open water. These systems provide high quality, low cost, very high spatial resolution (cm to m scale measurements) hyperspectral data in the nearshore environment, can be consistently flown at low altitude (minimizing atmospheric effects) below cloud cover and can be deployed on extremely short notice. With the continued advancement in sensor development, automated flight capabilities, and increased flight duration from vertical take off and landing (VTOL) platforms, water quality observation platforms such as these will soon be a common tool in resource manager's toolboxes.

The hyperspectral imaging system (HSI) developed by the NASA Glenn Research Center was used from 2015-2017 to collect high spatial resolution data over Lake Erie and the Ohio River. Paired with a vicarious correction approach implemented by the Michigan Tech Research Institute, radiance data collected by the HSI system can be converted to high quality reflectance data which can be used to generate near-real time (within 24 hours) products for the monitoring of harmful algal blooms using existing algorithms. The vicarious correction method relies on imaging a spectrally constant target to normalize HSI data for atmospheric and instrument calibration signals. A large asphalt parking lot near the Western Basin of Lake Erie was spectrally characterized and was determined to be a suitable correction target. Due to the HSI deployment aboard an aircraft, it is able to provide unique insights into water quality conditions not offered by space-based solutions. Aircraft can operate under cloud cover and flight paths can be chosen and changed on-demand, allowing for far more flexibility than space-based platforms. The HSI is also able to collect data at a high spatial resolution (~1 m), allowing for the monitoring of small water bodies, the ability to detect small patches of surface scum, and the capability to monitor the proximity of blooms to targets of interest such as water intakes. With this new rapid turnaround time, airborne data can serve as a complementary monitoring tool to existing satellite platforms, targeting critical areas and responding to bloom events on-demand.

Blooms of harmful cyanobacteria (cyanoHABs) have occurred on an annual basis in western Lake Erie for more than a decade. Previously, we developed and validated an algorithm to map the extent of the submerged and surface scum components of cyanoHABs using MODIS ocean-color satellite data. The algorithm maps submerged cyanoHABs by identifying high chlorophyll concentrations (>18 mg/m3) combined with water temperature >20 °C, while cyanoHABs surface scums are mapped using near-infrared reflectance values. Here, we adapted this algorithm for the SeaWiFS sensor to map the annual areal extents of cyanoHABs in the Western Basin of Lake Erie for the 20-year period from 1998 to 2017. The resulting classified maps were validated by comparison with historical in situ measurements, exhibiting good agreement (81% accuracy). Trends in the annual mean and maximum total submerged and surface scum extents demonstrated significant positive increases from 1998 to 2017. There was also an apparent 76% increase in year-to-year variability of mean annual extent between the 1998–2010 and 2011–2017 periods. The 1998–2017 time-series was also compared with several different river discharge nutrient loading metrics to assess the ability to predict annual cyanoHAB extents. The prediction models displayed significant relationships between spring discharge and cyanoHAB area; however, substantial variance remained unexplained due in part to the presence of very large blooms occurring in 2013 and 2015. This new multi-sensor time-series and associated statistics extend the current understanding of the extent, location, duration, and temporal patterns of cyanoHABs in western Lake Erie.

Lake Erie is a classic case of development, recovery from, and return to eutrophication, hypoxia, and harmful algal blooms. Forecast models are used annually to predict bloom intensity for the whole Western Lake Erie Basin, but do not necessarily reflect nearshore conditions or regional variations, which are important for local stakeholders. In this study we: 1) developed relationships between observed whole basin and nearshore bloom sizes, and 2) updated and extended a Bayesian seasonal bloom forecast model to provide new regional predictions. The western basin was subdivided into 5 km near-shore regions, and bloom start date, size, and intensity were quantified with MODIS-derived images of chlorophyll concentrations for July–October 2002–2016 for each subdivision and for the entire basin. While bloom severity within each subdivision is temporally and spatially unique, it increased over the study period in each subdivision. The models for the 5 km subdivisions explained between 83 and 95% of variability between regional sizes and whole bloom size for US subdivisions and 51% for the Canadian subdivision. By linking predictive basin-wide models to regional regression estimates, we are now able to better predict potential bloom impacts at scales and in specific areas that are vital to the economic well-being of the region and allow for better management responses.

The Kent State University (KSU) spectral decomposition method provides information about the spectral signals present in multispectral and hyperspectral images. Pre-processing steps that enhance signal to noise ratio (SNR) by 7.37–19.04 times, enables extraction of the environmental signals captured by the National Aeronautics and Space Administration (NASA) Glenn Research Center's, second generation, Hyperspectral imager (HSI2) into multiple, independent components. We have accomplished this by pre-processing of Level 1 HSI2 data to remove stripes from the scene, followed by a combination of spectral and spatial smoothing to further increase the SNR and remove non-Lambertian features, such as waves. On average, the residual stochastic noise removed from the HSI2 images by this method is 5.43 ± 1.42%. The method also enables removal of a spectrally coherent residual atmospheric bias of 4.28 ± 0.48%, ascribed to incomplete atmospheric correction. The total noise isolated from signal by the method is thus

Harmful algal blooms (HABs) have become a major health and environmental concern in the Great Lakes. In 2014, severe HABs prompted the State of Ohio to request NASA Glenn Research Center (GRC) to assist with monitoring algal blooms in Lake Erie. The most notable species of HAB is Microcystis aeruginosa, a hepatotoxin producing cyanobacteria that is responsible for liver complications for humans and other fauna that come in contact with these blooms. NASA GRC conducts semiweekly flights in order to gather up-to-date imagery regarding the blooms' spatial extents and concentrations. Airborne hyperspectral imagery is collected using two hyperspectral imagers, HSI-2 and HSI-3. Hyperspectral imagery is necessary in order to conduct experiments on differentiation of algal bloom types based on their spectral reflectance. In this analysis, imagery from September 19, 2016 was utilized to study the subpixel variability within the footprint of arbitrary sized pixels using several analysis techniques. This particular data set is utilized because it represents a worst case scenario where there is significant potential for public health concern due to high concentrations of microcystin toxin found in the water on this day and the concurrent observational challenges to accurately measure the algal bloom concentration variability with a remote sensing system due to the blooms high spatial variability. It has been determined that the optimal spatial resolution to monitor algal blooms in the Great Lakes is at most 50 m, and for much lower error 25 m, thus allowing for greater ease in identifying high concentration blooms near the surface. This resolution provides the best sensitivity to high concentration areas that are of significant importance in regard to human health and ecological damage.

Beetles (Coleoptera) are a diverse group of overlooked pollinators, considered particularly important in tropical ecosystems. The role of the most diverse beetle family, Staphylinidae, as pollinators is generally considered minor, yet their relationships with plants are mostly unknown. Although often referred to as opportunistic visitors, it is arguable that the true extent of rove beetle pollination is underestimated given their frequency of visitation to flowers. This review comprehensively analysed the plant–pollinator or visitor interactions of the Staphylinidae and uncovered 108 well-described staphylinid–flower interactions across 27 seed plant families. Of these interactions, Staphylinidae were considered either potential or conclusive pollinators for 56 plant species, having either a primary or secondary role in pollination. Conversely, Staphylinidae were visitors to 40 plant species with a negligible role in pollination. For the remaining 12 interactions and additional anecdotal reports, the role of staphylinids as pollinators was unresolved. Staphylinid–flower interactions were most prevalent in the monocots and magnoliids (families: Araceae, Annonaceae, Arecaceae, and Magnoliaceae) involving predominantly generalist pollination systems, and interactions were limited to six staphylinid subfamilies (Omaliinae, Tachyporinae, Aleocharinae, Oxytelinae, Paederinae, and Staphylininae). Trends in the involvement of staphylinid subfamilies with particular plant lineages were identified, associated with differences in insect habit and floral rewards. Overall this review indicates that the role of Staphylinidae as pollinators, and Coleoptera as a whole, is underestimated. Caution, however, must be given to inferring the role of staphylinids in pollination because rove beetles commonly function as inadvertent secondary pollinators or antagonists there to fulfil other ecological roles.