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All the major sixteenth-century Reformers knew something about the early church and used the early Fathers. As an Augustinian monk and professor of theology, however, Luther’s knowledge and use of the great Father was both deeper and more nuanced. While indebted to Augustine, Luther went further in defining what it meant for theology to be ‘scriptural’. He saw history as the interaction of God’s two regimes, and the church of every age as weak and flawed but conquering through the cross of Christ. This led him to a free use of the Fathers without being constrained to always agree with or imitate them. The comfort he received from the Apostles’ Creed in particular led him to appreciate the early creedal statements, and so it was natural for him to use them as models when formulating the new confessions required in his own day. The sixteenth-century heritage of written confessions of faith is a heritage under-appreciated but still vital for church bodies today.1

My colleagues thought Mr. Corman was unreasonable, maybe even dangerous. I didn't agree. [ABSTRACT FROM AUTHOR]

The use of pulsed electric fields (PEF) as a nonthermal technology for the decontamination of foods is of growing interest. This study aimed to enhance the inactivation of Escherichia coli, Listeria innocua, and Salmonella enterica in Gomori buffer using a combination of nsPEF and hydrogen peroxide (H2O2). Three sub-MIC concentrations (0.1, 0.3, and 0.5%) of H2O2 and various contact times ranging from 5-45 min were tested. PEF exposures as both single (1000 pulse) and split-dose (500+500 pulse) trains were delivered via square-wave, monopolar, 600 ns pulses at 21 kV/cm and 10 Hz. We demonstrate that >5 log CFU/mL reduction can be attained from combination PEF/H2O2 treatments with a 15 min contact time for E. coli (0.1%) and a 30 min contact time for L. innocua and S. enterica (0.5%), despite ineffective results from either individual treatment alone. A 5 log reduction in microbial population is generally the lowest acceptable level in consideration of food safety and represents inactivation of 99.999% of bacteria. Split-dose PEF exposures enhance lethality for several tested conditions, indicating greater susceptibility to PEF after oxidative damage has occurred., Competing Interests: The authors declare no competing interests., (Copyright: © 2024 Rosenzweig et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Deformation of the cell membrane is well understood from the viewpoint of protein interactions and free energy balance. However, the various dynamic properties of the membrane, such as lipid packing and hydrophobicity, and their relationship with cell membrane deformation are unknown. Therefore, the deformation of 1,2-dipalmitoyl- sn-glycero -3-phosphocholine (DPPC) and oleic acid (OA) giant unilamellar vesicles (GUVs) was induced by heating and cooling cycles, and time-lapse analysis was conducted based on the membrane hydrophobicity and physical parameters of "single-parent" and "daughter" vesicles. Fluorescence ratiometric analysis by simultaneous dual-wavelength detection revealed the variation of different hydrophilic GUVs and enabled inferences of the "daughter" vesicle composition and the "parent" membrane's local composition during deformation; the "daughter" vesicle composition of OA was lower than that of the "parents", and lateral movement of OA was the primary contributor to the formation of the "daughter" vesicles. Thus, our findings and the newly developed methodology, named in situ quantitative membrane property-morphology relation (QmPMR) analysis, would provide new insights into cell deformation and accelerate research on both deformation and its related events, such as budding and birthing.

Residential landscapes are essential to the sustainability of large areas of the United States. However, spatial and temporal variation across multiple domains complicates developing policies to balance these systems' environmental, economic, and equity dimensions. We conducted multidisciplinary studies in the Baltimore, MD, USA, metropolitan area to identify locations (hotspots) or times (hot moments) with a disproportionate influence on nitrogen export, a widespread environmental concern. Results showed high variation in the inherent vulnerability/sensitivity of individual parcels to cause environmental damage and in the knowledge and practices of individual managers. To the extent that hotspots are the result of management choices by homeowners, there are straightforward approaches to improve outcomes, e.g. fertilizer restrictions and incentives to reduce fertilizer use. If, however, hotspots arise from the configuration and inherent characteristics of parcels and neighborhoods, efforts to improve outcomes may involve more intensive and complex interventions, such as conversion to alternative ecosystem types., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

This study is the first to report the enhancement of cell migration and proliferation induced by in vitro microsecond pulsed electric field (μsPEF) exposure of primary bovine annulus fibrosus (AF) fibroblast-like cells. AF primary cells isolated from fresh bovine intervertebral disks (IVDs) are exposed to 10 and 100 μsPEFs with different numbers of pulses and applied electric field strengths. The results indicate that 10 μs-duration pulses induce reversible electroporation, while 100 μs pulses induce irreversible electroporation of the cells. Additionally, μsPEF exposure increased AF cell proliferation up to 150% while increasing the average migration speed by 0.08 μm/min over 24 h. The findings suggest that the effects of PEF exposure on cells are multifactorial-depending on the duration, intensity, and number of pulses used in the stimulation. This highlights the importance of optimizing the μsPEF parameters for specific cell types and applications. For instance, if the goal is to induce cell death for cancer treatment, then high numbers of pulses can be used to maximize the lethal effects. On the other hand, if the goal is to enhance cell proliferation, a combination of the number of pulses and the applied electric field strength can be tuned to achieve the desired outcome. The information gleaned from this study can be applied in the future to in vitro cell culture expansion and tissue regeneration., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Despite the abundance of tree diversity in the natural world, and generally high tree species richness in urban areas, urban forests continue to be dominated by a limited number of species. As socio-ecological systems, urban forests are shaped by historical and current management efforts and decision-making across a wide range of human actors. Drawing on past research, we offer a conceptual framework for describing the complex interactions among tree producers and consumers as trees are selected, grown, specified, and planted in private and public urban areas. We illustrate how multiple layers of selection criteria filter down the entirety of potential local tree diversity to a handful of commonly used and accepted tree species. We detail the actors and decision-makers who impact tree composition and diversity across several land types. Finally, we identify research, education, and outreach needs as they relate to creating more diverse and resilient urban forest ecosystems., (© 2023. The Author(s) under exclusive licence to Royal Swedish Academy of Sciences.)

Land-use change is highly dynamic globally and there is great uncertainty about the effects of land-use legacies on contemporary environmental performance. We used a chronosequence of urban grasslands (lawns) that were converted from agricultural and forested lands from 10 to over 130 years prior to determine if land-use legacy influences components of soil biodiversity and composition over time. We used historical aerial imagery to identify sites in Baltimore County, MD (USA) with agricultural versus forest land-use history. Soil samples were taken from these sites as well as from existing well-studied agricultural and forest sites used as historical references by the National Science Foundation Long-Term Ecological Research Baltimore Ecosystem Study program. We found that the microbiomes in lawns of agricultural origin were similar to those in agricultural reference sites, which suggests that the ecological parameters on lawns and reference agricultural systems are similar in how they influence soil microbial community dynamics. In contrast, lawns that were previously forest showed distinct shifts in soil bacterial composition upon recent conversion but reverted back in composition similar to forest soils as the lawns aged over decades. Soil fungal communities shifted after forested land was converted to lawns, but unlike bacterial communities, did not revert in composition over time. Our results show that components of bacterial biodiversity and composition are resistant to change in previously forested lawns despite urbanization processes. Therefore land-use legacy, depending on the prior use, is an important factor to consider when examining urban ecological homogenization., (© 2023. The Author(s).)

Pulsed electric fields (PEFs) are often used to pretreat foods to enhance subsequent processes, such as drying, where maintaining food product quality is important for consumer satisfaction. This study aims to establish a threshold PEF exposure to determine the doses at which electroporation is viable for use on spinach leaves, wherein integrity is maintained postexposure. Three numbers of consecutive pulses (1, 5, 50) and two pulse durations (10 and 100 μs) have been examined herein at a constant pulse repetition of 10 Hz and 1.4 kV/cm field strength. The data indicate that pore formation in itself is not a cause for loss of spinach leaf food quality, i.e., significant changes in color and water content. Rather, cell death, or the rupture of the cell membrane from a high-intensity treatment, is necessary to significantly alter the exterior integrity of the plant tissue. PEF exposures thus can be used on leafy greens up until the point of inactivation before consumers would see any alterations, making reversible electroporation a viable treatment for consumer-intended products. These results open up future opportunities to use emerging technologies based on PEF exposures and provide useful information in setting parameters to avoid food quality diminishment., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Electropermeabilization of biomembranes often is measured by microscopic imaging of a membrane-impermeable fluorophore that penetrates the cells following pulsed electric field (PEF) exposure. PEF exposure subsequently changes physiological properties of tissue. One way to probe these changes in tissue is measuring electrical properties by way of electrochemical impedance spectroscopy (EIS). In this study, we analyse impedance and conductivity of bovine annulus fibrosus (AF) cartilage before and after exposures to PEF of 100 μ s duration. Two PEF parameters-electric field amplitude and number of pulses-are varied, and total specific dose of PEF is calculated. AF tissue conductivity increases with both amplitude and number of pulses, indicating electropermeabilization of the AF cells. A Live/Dead cell imaging assay validates the EIS measurements, indicating intratissue cell permeabilization by μ sPEF exposure. These results support the extension of EIS to monitor extent of electropermeabilization of cells within cartilage tissue., (Creative Commons Attribution license.)

Electroporation-based technologies using microsecond pulsed electric field (µsPEF) exposures are established as laboratory and clinical tools that permeabilize cell membranes. We demonstrate a µsPEF bioeffect on nucleocytoplasmic import and export of enzymes that regulate genetic expression, histone deacetylases (HDAC) -4 and -5. Their μsPEF-induced nucleocytoplasmic transport depends on presence and absence of extracellular calcium ions (Ca 2+ ) for both MCF7 and CHO-K1 cells. Exposure to 1, 10, 30 and 50 consecutive square wave pulses at 1 Hz and of 100 µs duration with 1.45 kV/cm magnitude leads to translocation of endogenous HDAC4 and HDAC5. We posit that by eliciting a rise in intracellular Ca 2+ concentration, a signaling pathway involving kinases, such as Ca 2+ /CaM-dependent protein kinase II (CaMKII), is activated. This cascade causes nuclear export and import of HDAC4 and HDAC5. The potential of µsPEF exposures to control nucleocytoplasmic transport unlocks future opportunities in epigenetic modification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Safaei and Thompson.)

Thrombotic microangiopathy (TMA) is characterized by thrombocytopenia, microangiopathic haemolytic anaemia and end organ damage. TMAs have varying underlying pathophysiology and can therefore present with an array of clinical presentations. Renal involvement is common as the kidney is particularly susceptible to the endothelial damage and microvascular occlusion. TMAs require rapid assessment, diagnosis, and commencement of appropriate treatment due to the high morbidity and mortality associated with them. Ground-breaking research into the pathogenesis of TMAs over the past 20 years has driven the successful development of targeted therapeutics revolutionizing patient outcomes. This review outlines the clinical presentations, pathogenesis, diagnostic tests and treatments for TMAs., (© 2022 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Background: Decalcification and gingivitis caused by plaque accumulation around brackets are common iatrogenic effects of fixed appliances. The influence of conventional versus self-ligating bracket design on microbial colonisation is unknown., Objective: To assess the levels of microbial colonisation associated with conventional and self-ligating brackets., Search Sources: Three databases were searched for publications from 2009 to 2021., Data Selection: Randomised controlled trials comparing levels of microbial colonisation before and during treatment with conventional and self-ligating brackets were assessed independently and in duplicate., Data Extraction: Data were extracted independently by two authors from the studies that fulfilled the inclusion criteria. Risk of bias assessments were made using the revised Cochrane risk of bias tool for randomized trials. The quality of the included studies was assessed using the Critical Appraisal Skills Programme Checklist., Results: A total of 11 randomised controlled trials were included in this systematic review. Six of the studies were found to be at low risk of bias and five presented with some concerns. The studies were considered moderate to high quality. Five trials reported no statistically significant difference in microbial colonisation between bracket types. The remaining studies showed mixed results, with some reporting increased colonisation of conventional brackets and others increased colonisation of self-ligating brackets. The heterogeneity of study methods and outcomes precluded meta-analysis., Conclusion: Of the 11 studies included in this systematic review, five found no differences in colonisation between conventional and self-ligating brackets. The remaining studies showed mixed results. The evidence is inconclusive regarding the association between bracket design and levels of microbial colonisation.

Climate change-related soil salinization increases plant stress and decreases productivity. Soil microorganisms are thought to reduce salt stress through multiple mechanisms, so diverse assemblages could improve plant growth under such conditions. Previous studies have shown that microbiome selection can promote desired plant phenotypes, but with high variability. We hypothesized that microbiome selection would be more consistent in saline soils by increasing potential benefits to the plants. In both salt-amended and untreated soils, we transferred forward Brassica rapa root microbiomes (from high-biomass or randomly selected pots) across six planting generations while assessing bacterial (16S rRNA) and fungal (ITS) composition in detail. Uniquely, we included an add-back control (re-adding initial frozen soil microbiome) as a within-generation reference for microbiome and plant phenotype selection. We observed inconsistent effects of microbiome selection on plant biomass across generations, but microbial composition consistently diverged from the add-back control. Although salt amendment strongly impacted microbial composition, it did not increase the predictability of microbiome effects on plant phenotype, but it did increase the rate at which microbiome selection plateaued. These data highlight a disconnect in the trajectories of microbiomes and plant phenotypes during microbiome selection, emphasizing the role of standard controls to explain microbiome selection outcomes., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Recent advances in three-dimensional (3D) bioprinting technologies have enabled precise patterning of cellular components along with biomimetic constructs for tissue engineering and regenerative medicine. The viscoelasticity of bioinks regulate printability and the smallest feature size in 3D bioprinted constructs. The impact of cellular components is typically neglected when choosing 3D bioprinting parameters. In this short communication, we quantified the effect of cell densities on the printability of hydrogel bioinks. Unexpectedly, our results show that encapsulated cells reduced the steady shear viscosity of gelatin-based bioinks by approximately 50% and the minimum force for onset of flow by approximately 30%. These results may justify the lower spatial resolution in 3D bioprinted cell-laden hydrogels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca 2+ ) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca 2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca 2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca 2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca 2+ -free solution.

Cell swelling and blebbing has been commonly observed following nanosecond pulsed electric field (nsPEF) exposure. The hypothesized origin of these effects is nanoporation of the plasma membrane (PM) followed by transmembrane diffusion of extracellular fluid and disassembly of cortical actin structures. This investigation will provide evidence that shows passive movement of fluid into the cell through nanopores and increase of intracellular osmotic pressure are not solely responsible for this observed phenomena. We demonstrate that phosphatidylinositol-4,5-bisphosphate (PIP 2 ) depletion and hydrolysis are critical steps in the chain reaction leading to cellular blebbing and swelling. PIP 2 is heavily involved in osmoregulation by modulation of ion channels and also serves as an intracellular membrane anchor to cortical actin and phospholipase C (PLC). Given the rather critical role that PIP 2 depletion appears to play in the response of cells to nsPEF exposure, it remains unclear how its downstream effects and, specifically, ion channel regulation may contribute to cellular swelling, blebbing, and unknown mechanisms of the lasting "permeabilization" of the PM.

Biased agonism describes the ability of distinct G protein-coupled receptor (GPCR) ligands to stabilise distinct receptor conformations leading to the activation of different cell signalling pathways that can deliver different physiologic outcomes. This phenomenon is having a major impact on modern drug discovery as it offers the potential to design ligands that selectively activate or inhibit the signalling pathways linked to therapeutic effects with minimal activation or blockade of signalling pathways that are linked to the development of adverse on-target effects. However, the explosion in studies of biased agonism at multiple GPCR families in recombinant cell lines has revealed a high degree of variability on descriptions of biased ligands at the same GPCR and raised the question of whether biased agonism is a fixed attribute of a ligand in all cell types. The current study addresses this question at the mu-opioid receptor (MOP). Here, we have systematically assessed the impact of differential cellular protein complement (and cellular background), signalling kinetics and receptor species on our previous descriptions of biased agonism at MOP by several opioid peptides and synthetic opioids. Our results show that all these factors need to be carefully determined and reported when considering biased agonism. Nevertheless, our studies also show that, despite changes in overall signalling profiles, ligands that previously showed distinct bias profiles at MOP retained their uniqueness across different cell backgrounds., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Urban grasslands, landscapes dominated by turfgrasses for aesthetic or recreational groundcovers, are rapidly expanding in the United States and globally. These managed ecosystems are often less diverse than the natural or agricultural lands they replace, leading to potential losses in ecosystem functioning. Research in non-urban systems has provided evidence for increases in multiple ecosystem functions associated with greater plant diversity. To test if biodiversity-ecosystem function findings are applicable to urban grasslands, we examined the effect of plant species and genotypic diversity on three ecosystem functions, using grassland assemblages of increasing diversity that were grown within a controlled environment facility. We found positive effects of plant diversity on reduced nitrate leaching and plant productivity. Soil microbial diversity (Mean Shannon Diversity, H') of bacteria and fungi were also enhanced in multi-species plantings, suggesting that moderate increments in plant diversity influence the composition of soil biota. The results from this study indicate that plant diversity impacts multiple functions that are important in urban ecosystems; therefore, further tests of urban grassland biodiversity should be examined in situ to determine the feasibility of manipulating plant diversity as an explicit landscape design and function trait.

Differential regulation of the μ-opioid receptor (MOR), a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor, contributes to the clinically limiting effects of opioid analgesics, such as morphine. We used biophysical approaches to quantify spatiotemporal MOR signaling in response to different ligands. In human embryonic kidney (HEK) 293 cells overexpressing MOR, morphine caused a Gβγ-dependent increase in plasma membrane-localized protein kinase C (PKC) activity, which resulted in a restricted distribution of MOR within the plasma membrane and induced sustained cytosolic extracellular signal-regulated kinase (ERK) signaling. In contrast, the synthetic opioid peptide DAMGO ([d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin) enabled receptor redistribution within the plasma membrane, resulting in transient increases in cytosolic and nuclear ERK activity, and, subsequently, receptor internalization. When Gβγ subunits or PKCα activity was inhibited or when the carboxyl-terminal phosphorylation sites of MOR were mutated, morphine-activated MOR was released from its restricted plasma membrane localization and stimulated a transient increase in cytosolic and nuclear ERK activity in the absence of receptor internalization. Thus, these data suggest that the ligand-induced redistribution of MOR within the plasma membrane, and not its internalization, controls its spatiotemporal signaling., (Copyright © 2016, American Association for the Advancement of Science.)

Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Biased agonism is having a major impact on modern drug discovery, and describes the ability of distinct G protein-coupled receptor (GPCR) ligands to activate different cell signaling pathways, and to result in different physiologic outcomes. To date, most studies of biased agonism have focused on synthetic molecules targeting various GPCRs; however, many of these receptors have multiple endogenous ligands, suggesting that "natural" bias may be an unappreciated feature of these GPCRs. The μ-opioid receptor (MOP) is activated by numerous endogenous opioid peptides, remains an attractive therapeutic target for the treatment of pain, and exhibits biased agonism in response to synthetic opiates. The aim of this study was to rigorously assess the potential for biased agonism in the actions of endogenous opioids at the MOP in a common cellular background, and compare these to the effects of the agonist d-Ala2-N-MePhe4-Gly-ol enkephalin (DAMGO). We investigated activation of G proteins, inhibition of cAMP production, extracellular signal-regulated kinase 1 and 2 phosphorylation, β-arrestin 1/2 recruitment, and MOP trafficking, and applied a novel analytical method to quantify biased agonism. Although many endogenous opioids displayed signaling profiles similar to that of DAMGO, α-neoendorphin, Met-enkephalin-Arg-Phe, and the putatively endogenous peptide endomorphin-1 displayed particularly distinct bias profiles. These may represent examples of natural bias if it can be shown that they have different signaling properties and physiologic effects in vivo compared with other endogenous opioids. Understanding how endogenous opioids control physiologic processes through biased agonism can reveal vital information required to enable the design of biased opioids with improved pharmacological profiles and treat diseases involving dysfunction of the endogenous opioid system., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Unlabelled: Opioids, such as morphine, are the most clinically useful class of analgesic drugs for the treatment of acute and chronic pain. However, the use of opioids is greatly limited by the development of severe adverse side effects. Consequently, drug discovery efforts have been directed towards improving the therapeutic profile of opioid-based treatments. Opioid receptors are members of the family of GPCRs. As such, the recent GPCR paradigms of biased agonism and allosterism may provide novel avenues for more effective analgesics. Biased agonism (or functional selectivity) has been described for all the opioid receptor family members. Furthermore, the first allosteric modulators of opioid receptors have very recently been described. However, identification and quantification of biased agonism in a manner that is informative to medicinal chemists and drug discovery programmes still remains a challenge. In this review, we examine the progress, to date, towards identification and quantification of biased agonism and allosterism at the μ-opioid receptor in the context of its implications for the discovery of better and safer analgesics., Linked Articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2., (© 2014 The British Pharmacological Society.)

The interaction between nsPEF-induced Ca(2+) release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca(2+) the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca(2+) (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca(2+) is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca(2+) leads to a whole cell response. The reported observations of Ca(2+) dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this publication, we demonstrate that exposure of Jurkat and U937 cells to nanosecond pulsed electrical fields (nsPEF) can modulate the extrinsic-mediated apoptotic pathway via the Fas/CD95 death receptor. An inherent difference in survival between these two cell lines in response to 10 ns exposures has been previously reported (Jurkat being more sensitive to nsPEF than U937), but the reason for this sensitivity difference remains unknown. We found that exposure of each cell line to 100, 10 ns pulses at 50 kV/cm caused a marked increase in expression of cFLIP (extrinsic apoptosis inhibitor) in U937 and FasL (extrinsic apoptosis activator) in Jurkat, respectively. Measurement of basal expression levels revealed an inherent difference between U937 cells, having a higher expression of cFLIP, and Jurkat cells, having a higher expression of FasL. From these data, we hypothesize that the sensitivity difference between the cells to nsPEF exposure may be directly related to expression of extrinsic apoptotic regulators. To validate this hypothesis, we used siRNA to knockdown cFLAR (coding for cFLIP protein) expression in U937, and FasL expression in Jurkat and challenged them to 100, 10 ns pulses at 150 kV/cm, a typical lethal dose. We observed that U937 survival was reduced nearly 60% in the knockdown population while Jurkat survival improved ~40%. These findings support the hypothesis that cell survival following 10 ns pulse exposures depends on extrinsic apoptotic regulators. Interestingly, pretreatment of U937 with a 100-pulse, 50 kV/cm exposure (to amplify cFLAR expression) significantly reduced the lethality of a 150 kV/cm, 100-pulse exposure applied 24 h later. From these data, we conclude that the observed survival differences between cells, exposed to 10 ns pulsed electric fields, is due to inherent cell biochemistry rather than the biophysics of the exposure itself. Understanding cell sensitivity to nsPEF may provide researchers/clinicians with a predicable way to control or avoid unintended cell death during nsPEF exposure.

This review focuses on the existence and function of multiple endogenous agonists of the somatostatin and opioid receptors with an emphasis on their expression in the gastrointestinal tract. These agonists generally arise from the proteolytic cleavage of prepropeptides during peptide maturation or from degradation of peptides by extracellular or intracellular endopeptidases. In other examples, endogenous peptide agonists for the same G protein-coupled receptors can be products of distinct genes but contain high sequence homology. This apparent biological redundancy has recently been challenged by the realization that different ligands may engender distinct receptor conformations linked to different intracellular signaling profiles and, as such the existence of distinct ligands may underlie mechanisms to finely tune physiological responses. We propose that further characterization of signaling pathways activated by these endogenous ligands will provide invaluable insight into the mechanisms governing biased agonism. Moreover, these ligands may prove useful in the design of novel therapeutic tools to target distinct signaling pathways, thereby favoring desirable effects and limiting detrimental on-target effects. Finally we will discuss the limitations of this area of research and we will highlight the difficulties that need to be addressed when examining endogenous bias in tissues and in animals.

The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (<2  nm) are created in the plasma membrane in contrast to larger diameter pores (>2  nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2  kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.

Nanosecond pulsed electric fields (nsPEFs) perturb membranes of cultured mammalian cells in a dose-dependent manner with different types of cells exhibiting characteristic survivability. Adherent cells appear more robust than non-adherent cells during whole-cell exposure. We hypothesize that cellular elasticity based upon the actin cytoskeleton is a contributing parameter, and the alteration of a cell's actin cortex will significantly affect viability upon nsPEF exposure. Chinese hamster ovary (CHO) cells that are (a) untreated, (b) treated with latrunculin A to inhibit actin polymerization, or (c) exposed to nsPEFs have been probed using atomic force microscopy (AFM) force-indentations. Exposure to 50 or 100 pulses of 10 ns duration and 150 kV/cm in a single dosage approximately lowers average CHO cell elastic modulus by half, whereas latrunculin lowers it more than 75%. Latrunculin pre-treatment disrupts the actin cortex enough that it negates cumulative damage by equally fractionated (i.e., two rounds of 50 pulses each, separated by 10 min) dosages of nsPEFs as seen in untreated and dimethyl sulfoxide (DMSO)-treated cells with propidium uptake, phosphatidylserine externalization, and 24 h viability according to MTT and CellTiter Glo assays. These results suggest a correlation among cell stiffness, cytoskeletal integrity, and susceptibility to recurrent exposures to nsPEFs, which emphasizes a mechanobiological underpinning of nsPEF bioeffects., (© 2014 Wiley Periodicals, Inc.)

Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways--phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2 kV/cm, 600 ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions., (Published by Elsevier B.V.)

Background: Working in hospice care is a highly challenging yet rewarding profession. However, the challenges of working with dying patients and their families can overwhelm even the most highly dedicated professional, leading to burnout, compassion fatigue, anxiety, and depression., Objective: The aim of this study was to better understand how stress affects the mental health of hospice workers in terms of burnout and compassion fatigue and how they cope with these issues., Methods: Data for this study are from Compassion Fatigue and You, a cross-sectional survey of hospice staff from across Minnesota. We surveyed 547 hospice workers throughout Minnesota to better understand the overall mental health of staff, including levels of stress, burnout, and compassion fatigue, and how they cope with these issues. The study was conducted in 2008 and 2009 through a private, not-for-profit research institute affiliated with a large Midwestern health plan., Results: Hospice staff reported high levels of stress, with a small but significant proportion reporting moderate-to-severe symptoms of depression, anxiety, compassion fatigue, and burnout. Staff reported managing their stress through physical activity and social support, and they suggested that more opportunities to connect with coworkers and to exercise could help decrease staff burnout., Conclusions: Poor mental health places staff at risk for burnout and likely contributes to staff leaving hospice care; this is a critical issue as the profession attempts to attract new staff to meet the expanding demands for hospice care.

The persistent influx of ions through nanopores created upon cellular exposure to nanosecond pulse electric fields (nsPEF) could be used to modulate neuronal function. One ion, calcium (Ca(2+)), is important to action potential firing and regulates many ion channels. However, uncontrolled hyper-excitability of neurons leads to Ca(2+) overload and neurodegeneration. Thus, to prevent unintended consequences of nsPEF-induced neural stimulation, knowledge of optimum exposure parameters is required. We determined the relationship between nsPEF exposure parameters (pulse width and amplitude) and nanopore formation in two cell types: rodent neuroblastoma (NG108) and mouse primary hippocampal neurons (PHN). We identified thresholds for nanoporation using Annexin V and FM1-43, to detect changes in membrane asymmetry, and through Ca(2+) influx using Calcium Green. The ED50 for a single 600 ns pulse, necessary to cause uptake of extracellular Ca(2+), was 1.76  kV/cm for NG108 and 0.84  kV/cm for PHN. At 16.2  kV/cm, the ED50 for pulse width was 95 ns for both cell lines. Cadmium, a nonspecific Ca(2+) channel blocker, failed to prevent Ca(2+) uptake suggesting that observed influx is likely due to nanoporation. These data demonstrate that moderate amplitude single nsPEF exposures result in rapid Ca(2+) influx that may be capable of controllably modulating neurological function.

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of bacteria of different types in pure water. Here, the BEPFM method is performed for the first time on physiologically relevant electrolyte media, such as Dulbecco's phosphate-buffered saline (DPBS) and Dulbecco's modified Eagle's medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria in DPBS are demonstrated. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media.

Harnessing electrical bias-induced mechanical motion on the nanometer and molecular scale is a critical step toward understanding the fundamental mechanisms of redox processes and implementation of molecular electromechanical machines. Probing these phenomena in biomolecular systems requires electromechanical measurements be performed in liquid environments. Here we demonstrate the use of band excitation piezoresponse force microscopy for probing electromechanical coupling in amyloid fibrils. The approaches for separating the elastic and electromechanical contributions based on functional fits and multivariate statistical analysis are presented. We demonstrate that in the bulk of the fibril the electromechanical response is dominated by double-layer effects (consistent with shear piezoelectricity of biomolecules), while a number of electromechanically active hot spots possibly related to structural defects are observed.

Ratings were collected from 102 native speakers of Spanish on the subjective frequency of occurrence of 330 Spanish words, including 120 deverbal compounds and their constituents. These ratings were found to be highly reliable, whether items were analyzed together or separately by type (i.e., compounds, nouns, verbs), as evidenced by indexes of internal consistency and test-retest reliability that were equal to or greater than .98. The validity of the normative ratings was attested to by statistically significant correlations with objective frequency, estimated at .63 for all items together, and .41, .51, and .78 for compounds, nouns, and verbs, respectively. Among the substantive issues addressed was the potential dependency in ratings for compounds and their associated verb-noun constituents. No relationship was discerned, supporting the idea that compound and constituent ratings are statistically independent in this experimental task. The theoretical and methodological implications of the findings are discussed. The ratings can be downloaded from http://brm.psychonomic-journals.org/content/supplemental.

Functional recognition imaging in scanning probe microscopy (SPM) using artificial neural network identification is demonstrated. This approach utilizes statistical analysis of complex SPM responses at a single spatial location to identify the target behavior, which is reminiscent of associative thinking in the human brain, obviating the need for analytical models. We demonstrate, as an example of recognition imaging, rapid identification of cellular organisms using the difference in electromechanical activity over a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method.

The internal validity of several types of experiments in experimental psychology and neuroscience depends in part on the possibility of controlling or manipulating critical lexical variables such as word frequency of occurrence. Two ways of estimating this variable are (1) objective frequency counts and (2) subjective ratings of word frequency. Each method produces estimates that generally agree (i.e., they are highly correlated) but that disagree substantially concerning the relative frequency of a number of words. To investigate this issue more closely, the global and local agreement of subjective frequency estimates was examined in detail for a pool of 6,202 words drawn from the OMNILEX database of French words (Desrochers, 2006; www.omnilex.uottawa.ca). The results indicated that objective and subjective frequencies are strongly correlated, subjective frequencies share a significant amount of bias variance with other lexical characteristics (e.g., imageability), and the codeterminants of subjective frequency are in an antagonistic relationship with one another. The implications of these results for the selection of lexical stimuli are discussed, and multiple variables to aid in item selection are reported. Supplemental materials for this study may be downloaded from brm.psychonomic-journals.org/content/supplemental.

Subjective frequency and imageability estimates for a sample of 3,600 French nouns were collected from two independent groups of 72 young adults each. Both groups received standard instructions and provided their ratings on a 7-point scale. The timing, sequencing, presentation of lexical stimuli, and recording of responses were controlled by a computer. All estimates of internal consistency and test-retest reliability (> or =.98) confirm the high level of precision and reliability of the ratings. Correlations with ratings drawn from similar studies were found to be positive and significant for subjective frequency (r > or = .85) and for imageability (r > or = .69). Subjective frequency was positively and significantly correlated with objective frequency estimates drawn from 10 different sources (r > or = .42). Subjective frequency and imageability were significantly correlated (r = .26), a relationship that was driven primarily by a sudden drop in imageability ratings for words with a subjective frequency rating below 2.5. The methodological implications of these findings are discussed. The ratings can be downloaded as supplemental materials from brm.psychonomic-journals.org/content/supplemental.

A standard part of vitrectomy surgery is to inject a bubble of gas into the eye, and an important question for the surgeon and patient alike is to know the volume of the bubble and how long it is likely to last. A related question of considerable importance is the mechanism by which it is absorbed. In this paper, we show how to use patients' measurements of the daily variation of their far points to calculate the changing volume of the bubble and hence the rate of gas absorption throughout the post-operative period. The results show that the rate of absorption contains a term in time squared, indicative of absorption through the exposed surface area of the aqueous fluid rather than via the exposed retina.

Electromechanical coupling is ubiquitous in biological systems, with examples ranging from simple piezoelectricity in calcified and connective tissues to voltage-gated ion channels, energy storage in mitochondria, and electromechanical activity in cardiac myocytes and outer hair cell stereocilia. Piezoresponse force microscopy (PFM) originally emerged as a technique to study electromechanical phenomena in ferroelectric materials, and in recent years has been employed to study a broad range of non-ferroelectric polar materials, including piezoelectric biomaterials. At the same time, the technique has been extended from ambient to liquid imaging on model ferroelectric systems. Here, we present results on local electromechanical probing of several model cellular and biomolecular systems, including insulin and lysozyme amyloid fibrils, breast adenocarcinoma cells, and bacteriorhodopsin in a liquid environment. The specific features of PFM operation in liquid are delineated and bottlenecks on the route towards nanometre-resolution electromechanical imaging of biological systems are identified.

This study reports the effect of exposure to liquid carbon dioxide on the mechanical properties of selected medical polymers. The tensile strengths and moduli of fourteen polymers are reported. Materials were exposed to liquid CO(2), or CO(2) + trace amounts of aqueous H(2)O(2), at 6.5 MPa and ambient temperature. Carbon dioxide uptake, swelling, and distortion were observed for the more amorphous polymers while polymers with higher crystallinity showed little effect from CO(2) exposure. Changes in tensile strength were not statistically significant for most plastics, and most indicated good tolerance to liquid CO(2). These results are relevant to evaluating the potential of liquid CO(2)-based sterilization technology.

Purpose: Previous studies demonstrated a negative correlation between prostate volume and biopsy yield. By decreasing prostate volume 5alpha-reductase inhibitors may enhance cancer detection, which may explain the greater detection of high grade tumors in the finasteride arm of the Prostate Cancer Prevention Trial., Materials and Methods: A mathematical model was constructed to analyze the effects of prostate and tumor volumes, and biopsy core number on cancer detection. The effects of the volume reduction observed with finasteride in the Prostate Cancer Prevention Trial were also modeled, as was the potential reduction in tumor volume needed to explain the observed difference in prostate cancer detection. The model was also applied to the Reduction by Dutasteride of Prostate Cancer Events study., Results: A higher number of biopsies are required to ensure a detection probability of 0.90 or greater in larger glands or with smaller tumors. In the Prostate Cancer Prevention Trial for a tumor volume of 1 cc a 17% increase in the detection rate in the finasteride arm would be predicted if there was no change in tumor volume, likewise the rate would be 11% to 17% for the dutasteride arm of the Reduction by Dutasteride of Prostate Cancer Events study. The calculated reduction in tumor volume needed to explain the difference in cancer detection between the finasteride and placebo arms of the Prostate Cancer Prevention Trial would be 51% to 66%., Conclusions: This model provides guidance on the optimal number of biopsy cores that accord with an earlier model. These findings also suggest that, if there were no reduction in tumor volume, 5alpha-reductase inhibitor therapy could lead to excess cancer detection, including high grade tumors.

Sophisticated univariate outlier screening procedures are not yet available in widely used statistical packages such as SPSS. However, SPSS can accept user-supplied programs for executing these procedures. Failing this, researchers tend to rely on simplistic alternatives that can distort data because they do not adjust to cell-specific characteristics. Despite their popularity, these simple procedures may be especially ill suited for some applications (e.g., data from reaction time experiments). A user friendly SPSS Production Facility implementation of the shifting z score criterion procedure (Van Selst & Jolicoeur, 1994) is presented in an attempt to make it easier to use. In addition to outlier screening, optional syntax modules can be added that will perform tedious database management tasks (e.g., restructuring or computing means).

Two novels were selected from two distinct periods in John Steinbeck's career, and text samples from each were pooled according to era. Using quantitative measures of text emotion, between-sample and norm-relative comparisons were computed to evaluate a priori expectations regarding differences in the use of implicit emotional information. These hypotheses reflected abstract characteristics that transcend the focus of a single novel. Analysis of word-based measures did not yield the between-sample differences expected. Rather, the hypotheses were supported by distinct patterns of phoneme distribution. The findings confirm and extend previous work by demonstrating that authors of prose manipulate the emotional quality of phonemes, and that the associated patterns can occur at a more abstract level than previously demonstrated.

Two Santa Gertrudis cattle from a herd of 105 aborted within a 24-hour period. Bacteroides fragilis was isolated from tissues of each aborted fetus. Histopathologic lesions included placentitis and bronchopneumonia in which gram-negative, rod-shaped organisms were visible. The diagnostic workup failed to reveal other causes of abortion. Anaerobes are rarely implicated in bovine abortions, and no other report was found that described abortion in cattle due to B. fragilis.

Using data on death certificates, we examined age-adjusted cancer mortality rates for West Virginia men and women from 1980-1994. The leading causes of cancer deaths among men in the state in 1994 were cancers of the lung, prostate, colon, and pancreas, and non-Hodgkin's lymphoma. Among West Virginia women in 1994, cancer deaths were most often due to cancers of the lung, breast, colon, pancreas, and ovary. Based on polynomial regression analyses, many of the leading cancer mortality rates significantly increased during 1980-1994. The most striking increase was a 76% rise in lung cancer mortality among West Virginia women. These mortality data underscore the continuing need for tobacco control and other cancer control practices. Even though barriers to medical care challenge the state's health care professionals, the burden of cancer in West Virginia can be reduced by prevention, early detection, and appropriate treatment.