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Human population dynamics and their drivers are not well understood, especially over the long term and on large scales. Here, we estimate demographic growth trajectories from 9 to 3 ka BP across the entire globe by employing summed probability distributions of radiocarbon dates. Our reconstruction reveals multicentennial growth cycles on all six inhabited continents, which exhibited matching dominant frequencies and phase relations. These growth oscillations were often also synchronised with multicentennial variations in solar activity. The growth cycle for Europe, reconstructed based on  >91,000 radiocarbon dates, was backed by archaeology-derived settlement data and showed only a weak correlation with mean climate states, but a strong correlation with the stability of these states. We therefore suggest a link between multicentennial variations in solar activity and climate stability. This stability provided more favourable conditions for human subsistence success, and seems to have induced synchrony between regional growth cycles worldwide., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Few post-graduate training programs offer a comprehensive curriculum that includes structured clinical experiences to teach interprofessional care. To address this need, the United States Department of Veterans Affairs, Office of Academic Affiliations funded the Centers of Excellence in Primary Care Education (CoEPCE) from 2011-2019 to provide interprofessional curricula for health profession trainees (HPTs), including physician residents, nurse practitioner residents, pharmacy residents, and psychology residents. We examined changes over time in curricular domains, system impacts, and program practices based on HPT survey data and the qualitative evaluation of narrative feedback. An annual survey was administered to participants. Indirect standardized ratios were calculated for interprofessional professional education (IPE) program domains, system impacts, and program practices. Qualitative responses were coded based on curricular domains and key program components. The study cohort included 369 HPTs. Site and profession standardized indirect ratios across all professions indicated improvements in curricular domains, system impacts, and program practices, with significant differences observed for associated health HPTs as compared to other HPTs for performance improvement. Qualitative data indicated that profession was associated with differences in perceptions of the curriculum. Although improvements occurred over time, our findings support the need for the thoughtful consideration of profession-specific identity characteristics when designing interprofessional curricula.

This paper describes the Centers of Excellence in Primary Care Education (CoEPCE), a seven-site collaborative project funded by the Office of Academic Affiliations (OAA) within the Veterans Health Administration of the United States Department of Veterans Affairs (VA). The CoEPCE was established to fulfill OAA's vision of large-scale transformation of the clinical learning environment within VA primary care settings. This was accomplished by funding new Centers within VA facilities to develop models of interprofessional education (IPE) to teach health professions trainees to deliver high quality interprofessional team-based primary care to Veterans. Using reports and data collected and maintained by the National Coordinating Center over the first six years of the project, we describe program inputs, the multicomponent intervention, activities undertaken to develop the intervention, and short-term outcomes. The findings have implications for lessons learned that can be considered by others seeking large-scale transformation of education within the clinical workplace and the development of interprofessional clinical learning environments. Within the VA, the CoEPCE has laid the foundation for IPE and collaborative practice, but much work remains to disseminate this work throughout the national VA system.

DNP faculty who mentor students in quality improvement (QI) DNP projects often lack essential knowledge of QI principles. The purpose of this article is to guide DNP programs in developing confident and competent faculty mentors for QI DNP projects, facilitating DNP student success. At a multi-campus practice- and research-intensive university, strategies employed to teach College of Nursing faculty essential knowledge of QI principles comprise structural and process components. Structural supports standardize faculty workload, promote potential for collaborative scholarship, and provide instructional and resource support for faculty mentors. Organizational processes facilitate identification of practice sites and meaningful projects. A College of Nursing and university Institutional Review Board collaboration established policy to guide human subjects protection regarding DNP project activity, streamlining and standardizing the process. Library support mechanisms, access to ongoing faculty QI training and resources, and faculty feedback processes to improve QI faculty development are ongoing and sustained. Peer coaching provides continued support for faculty development. Initial process outcomes indicate that implemented strategies are well-received by faculty. The transition to competency-based education provides opportunities to create tools to measure multiple student quality and safety competencies highlighted in Domain 5 of The Essentials: Core Competencies for Professional Nursing Education and inform future directions for faculty development essential to support student success., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mixotrophic dinoflagellates (MTD) are a diverse group of organisms often responsible for the formation of harmful algal blooms. However, the development of dinoflagellate blooms and their effects on the plankton community are still not well explored. Here we relate the species succession of MTD with parallel changes of phytoplankton size spectra during periods of MTD dominance. We used FlowCAM analysis to acquire size spectra in the range 2-200 μm every one or two weeks from July to December 2007 at Helgoland Roads (Southern North Sea). Most size spectra of dinoflagellates were bimodal, whereas for other groups, e.g. diatoms and autotrophic flagellates, the spectra were unimodal, which indicates different resource use strategies of autotrophs and mixotrophs. The biomass lost in the size spectrum correlates with the potential grazing pressure of MTD. Based on size-based analysis of trophic linkages, we suggest that mixotrophy, including detritivory, drives species succession and facilitates the formation of bimodal size spectra. Bimodality in particular indicates niche differentiation through grazing of large MTD on smaller MTD. Phagotrophy of larger MTD may exceed one of the smaller MTD since larger prey was more abundant than smaller prey. Under strong light limitation, a usually overlooked refuge strategy may derive from detritivory. The critical role of trophic links of MTD as a central component of the plankton community may guide future observational and theoretical research., (© The Author(s) 2022. Published by Oxford University Press.)

Background: Doctoral-prepared nurses with diverse skillsets are required to meet nursing care needs in a complex and changing healthcare environment. A better understanding of the roles of doctoral-prepared nurses in Veterans Health Administration (VHA) workforce will help leverage their expertise to meet the needs of Veterans., Purpose: Assess the current roles of doctoral-prepared nurses within the VHA., Method: A cross-sectional survey was used to collect information on doctoral-prepared nurses within the VHA in 2016. Multiple strategies were used to identify doctoral-prepared nurses to recruit for an online survey. Survey invitations were sent electronically to unique individuals identified (N = 2403)., Results: Responses were received from 1015 nurses (42.2% response rate), with 929 nurses with a doctoral degree identified. DNP/DNAP degrees were most common (55%), followed by a PhD or DNS (33%). Significant differences were noted between nurses in different doctoral education categories across four main roles: research, clinical, educational, and administration., Conclusions: This survey generated the first comprehensive list of VHA doctoral-prepared nurses. Findings are being used by the VHA Office of Nursing Services to align degree types, duties of different positions, functional statements, and position expectations. Results support the continued need for collaboration between nurses with PhDs and DNPs to achieve research and clinical goals., (Published by Elsevier Inc.)

One of the most striking patterns at the land-ocean interface is the massive increase of chlorophyll-a (CHL) from continental shelves towards the coast, a phenomenon that is classically linked to physical features. Here I propose that the coastal-offshore CHL gradient in a shallow sea has biological origins related to phytoplankton mortality that are neglected in state-of-the-art biogeochemical models. I integrate a trait-based ecosystem model into a modular coupling framework that is applied to the southern North Sea (SNS). The coupled model very well reproduces daily, seasonal and inter-annual (2000-2014) dynamics and meso-scale patterns in macronutrients, zooplankton biomass, and CHL as observed in situ and by remote sensors. Numerical experiments reveal that coast-offshore CHL gradients may predominantly arise from a trophic effect as resolved by an increase in carnivorous grazing towards shallow waters. This carnivory gradient reflects higher near-coast abundance of juvenile fish and benthic filter feeders. Furthermore, the temporal evolution of CHL can be much affected by viral infection as a fast-responding loss process at intermediate to high phytoplankton concentrations. Viral control in the model also prevents excessive and unrealistic blooms during late spring. Herbivores as often only ecological factor considered for explaining the spatio-temporal phytoplankton distribution are in this study supplemented by pathogens as well as pelagic and benthic carnivores as powerful agents, which are barely represented in current modeling but can mediate physical drivers of coastal ecosystems., Competing Interests: The author has declared that no competing interests exist.

Background: Nurse Practitioner (NP) Postgraduate Residency programs are rapidly expanding. Currently, little is known about trainees' self-perceptions during these experiences., Purpose: Describe NP residents' perceptions of their strengths, areas for improvement, and goals while participating in the Veterans Affairs Centers of Excellence in Primary Care Education NP Residency program., Methods: NP residents responded to open-ended questions at three time points across their training year. Responses were analyzed using inductive and deductive approaches., Findings: NP residents self-reported strengths in patient-centered care and interprofessional teamwork. They identified clinical skill acquisition as the major area for improvement. Their short- and long-term goals focused on personal and professional growth., Discussion: These results suggest NPs prioritize clinical skill acquisition during a primary care residency. In contrast, leadership and performance improvement skills did not capture their attention. When aggregated at the programmatic level, assessments identified opportunities to improve the NP Residency program curriculum., (Published by Elsevier Inc.)

Aim: This article describes a job-shadowing project that partnered second-year medical and third-year pharmacy students with an advanced practice nurse (APN) for a four-hour job- shadowing experience., Background: In order to address the Interprofessional Education Collaborative (IPEC) Expert Panel core competencies of interprofessional communication, teamwork, and roles/responsibilities, this project implemented a job-shadowing experience to increase students' knowledge of APN roles and interprofessional collaborative team practices., Methods: Forty volunteer medical and pharmacy students were paired together and completed the job-shadowing activity with an APN. Assessment of knowledge was measured by pre- and post-project surveys., Results: Pre- and post-job-shadowing differences demonstrated statistical significance in the interprofessional domains of role awareness, collaboration and communication. These results suggest that an APN job-shadowing experience is effective in developing medical and pharmacy students' competencies in interprofessional collaborative practice., Conclusion: Specific recommendations include creating enhanced job-shadowing experiences within the curriculums of medicine, pharmacy, and nursing students, and assessing for evidence of enhanced IPEC competencies as a result of these learning experiences., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The Institute of Medicine has recommended the establishment of residency programs for advanced practice nursing graduates. Currently, the evidence about program effectiveness is limited., Purpose: To describe the nurse practitioner (NP) resident outcomes on seven competency domains established by the VA Centers of Excellence in Primary Care Education (VA CoEPCE)., Methods: We evaluated mean NP resident competency self-ratings and mean mentor ratings over the 12-month program across NP residency programs at five sites. Highest and lowest rated items and differences between NP resident self-ratings and mentor ratings were analyzed., Results: Mean NP resident self-ratings and mean mentor ratings demonstrated statistically significant improvement in all domains (p < .0001). At 12 months, NP residents were rated by their mentors as able to practice without supervision in all competency domains. At 1 and 12 months, clinical, leadership and quality improvement/population management competencies were the lowest scored domains while patient-centered care, interprofessional team collaboration, shared decision-making and sustained relationships competencies were highest., Conclusions: These results provide initial evidence for the effectiveness of VA CoEPCE NP residency programs and also highlight areas of needed improvement., (Published by Elsevier Inc.)

Health systems around the United States are embracing new models of primary care using interprofessional team-based approaches in pursuit of better patient outcomes, higher levels of satisfaction among patients and providers, and improved overall value. Less often discussed are the implications of new models of care for health professions education, including education for physicians, nurse practitioners, physician assistants, and other professions engaged in primary care. Described here is the interaction between care transformation and redesign of health professions education at the largest integrated delivery system in the United States: the Veterans Health Administration (VA). Challenges and lessons learned are discussed in the context of a demonstration initiative, the VA Centers of Excellence in Primary Care Education. Five sites, involving VA medical centers and their academic affiliates in Boise, Cleveland, San Francisco, Seattle, and West Haven, introduced interprofessional primary care curricula for resident physicians and nurse practitioner students beginning in 2011. Implementation struggles largely revolved around the operational logistics and cultural disruption of integrating educational redesign for medicine and nursing and facilitating the interface between educational and clinical activities. To realize new models for interprofessional teaching, faculty, staff, and trainees must understand the histories, traditions, and program requirements across professions and experiment with new approaches to achieving a common goal. Key recommendations for redesign of health professions education revolve around strengthening the union between interprofessional learning, team-based practice, and high-value care.

To integrate health care professional learners into patient-centered primary care delivery models, the Department of Veterans Affairs has funded five Centers of Excellence in Primary Care Education (CoEPCEs). The main goal of the CoEPCEs is to develop and test innovative structural and curricular models that foster transformation of health care training from profession-specific "silos" to interprofessional, team-based educational and care delivery models in patient-centered primary care settings. CoEPCE implementation emphasizes four core curricular domains: shared decision making, sustained relationships, interprofessional collaboration, and performance improvement. The structural models allow interprofessional learners to have longitudinal learning experiences and sustained and continuous relationships with patients, faculty mentors, and peer learners. This article presents an overview of the innovative curricular models developed at each site, focusing on nurse practitioner (NP) education. Insights on transforming NP education in the practice setting and its impact on traditional NP educational models are offered. Preliminary outcomes and sustainment examples are also provided., (Published by Mosby, Inc.)

Central to theoretical studies of trophic interactions is the formulation of the consumer response to varying food availability. Response functions, however, are only rarely derived in mechanistic ways. As a consequence, the uncertainty in the functional representation of feeding remains large, as, e.g., evident from the ongoing debate on the usage of Ivlev, or Holling type I, II, and III functions in aquatic ecosystem models. Here, I refer to the work of Sjöberg in Ecol Model 10:215-225 (1980) who proposed to apply elements of the queuing theory developed in operational research to plankton-plankton interactions. Within this frame, food item processing is subdivided into two major stages which may operate with variable synchronicity. Asynchronous phasing of the two stages enhances the probability of long total processing times. This phenomenon is here termed feeding intermittency. Intermittency is assumed to determine the functional form of grazing kinetics, for which a novel grazing function containing a "shape" parameter is derived. Using this function, I evaluate the hypotheses that intermittency is influenced by (1) patchiness in the prey field (e.g., related to turbulence), and (2) the ratio of actual prey size to optimal prey size. Evidence for the first hypothesis arises from explaining reported variations in clearance rates of Acartia tonsa under different turbulence regimes. Further model applications to ingestion data for rotifers, copepods, and ciliates support the view that an increasing food size enhances intermittency and, this way, affects functional grazing responses. In the application to ciliate grazing, a possible prey density effect appears, possibly due to an intermittent activation of a feeding sub-stage. Queueing theory offers mechanistic explanations for transitions between Holling I-, II-, and Ivlev-type grazing. In doing so for variable prey size ratios, it may also refine size-based ecosystem models which are increasingly emerging in plankton ecology.

As a rational basis for addressing both ecological and economic consequences of oil spills, a combination of simulating and estimating methods is proposed in this paper. An integration of the state-of-the-art oil spill contingency simulation system OSCAR with economic assessment method leads to realistic oil spill scenarios including their biological and economic impacts and the effort taken for combat as well as to an estimate for the total oil spill costs. In order to derive a simple function of total costs depending on few spill characteristics such as size, a number of hypothetical scenarios are simulated and evaluated for the German North Sea area. Results reveal that response costs of per unit oil spilled as well as integrated costs of oil released are simply characterized as two particular power-law functions of spill size. Such relationships can be straightforward transferred into decision making for efficient prevention and combat strategy in the study area.

In many coastal regions oil spills can be considered to be one of the most important risks for the coastal environment. Efficient contingency management in responding to oil spills is critically important. Strategic priorities in contingency management highly depend upon the importance attributed to different economic and ecological resources such as beaches or birds. Due to the lack of a market for natural resources in the real world, these resources cannot be directly measured in monetary terms. This increases the risk that natural resources and their services are neglected in contingency decision making. This paper evaluates these natural resources in a hypothetical market by using the methodology of stated choice experiments. Results from a pilot survey show that according to the perspective of individuals, an oil spill combat process should focus on the protection of coastal waters, beaches and eider ducks.

The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.

Photolon is a photosensitiser with demonstrated potential as an anti-tumour agent. In this study, an in vitro investigation was performed to determine the mechanism of Photolon-induced cell death. Cell killing was observed in a light-dependent manner and light-activated Photolon resulted in a significant production of reactive oxygen species (ROS), which could be blocked by type I ROS scavengers. Inhibition of ROS production using Trolox prevented Photolon-induced cell death. Light-activated Photolon caused no increase in caspase-3/7 activity, but a rapid increase in lactate dehydrogenase (LDH) release suggesting a loss of membrane integrity and subsequent cell death by necrosis. We conclude that the mechanism of Photolon-induced cell death involves the induction of ROS via a type I mechanism, which is ultimately responsible for cell killing by necrosis.

Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein beta (PI-TPbeta, SPIbeta cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIbetaS262A cells overexpressing a mutant PI-TPbeta that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of Ser-262 in PI-TPbeta is involved in the regulation of apoptosis. Conditioned medium (CM) from wtNIH3T3 cells contains bioactive factors, presumably arachidonic acid metabolites [H. Bunte, et al., 2006; M. Schenning, et al., 2004] that are able to protect SPIbeta cells against UV-induced apoptosis. CM from SPIbeta cells lacks this protective activity. However, after heat denaturation CM from SPIbeta cells regains a protective activity comparable with that of wtNIH3T3 cells. This indicates that CM from SPIbeta cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIbetaS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIbeta cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIbeta cells. In contrast, CM from wtNIH3T3 and SPIbetaS262A cells or heat-treated CM from SPIbeta cells does induce these anti-apoptotic markers. Since we have previously shown that some of the arachidonic acid metabolites present in CM from wtNIH3T3 cells are prostaglandin (PG) E(2) and PGF(2alpha), we investigated the effect of these PGs on cell survival. Although PGE(2) and PGF(2alpha) were found to protect wtNIH3T3 and SPIbetaS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIbeta cells. The fact that the concentrations of PGE(2) and PGF(2alpha) in the CM from SPIbeta cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIbeta cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE(2) and PGF(2alpha), an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIbetaS262A cells but not in SPIbeta cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIbeta cells.

Since their discovery and subsequent purification from mammalian tissues more than 30 years ago an impressive number of studies have been carried out to characterize and elucidate the biological functions of phosphatidylcholine transfer protein (PC-TP), phosphatidylinositol transfer protein (PI-TP) and non-specific lipid transfer protein, more commonly known as sterol carrier protein 2 (SCP-2). Here I will present information to show that these soluble, low-molecular weight proteins constitute domain structures in StArR-related lipid transfer (START) proteins (i.e. PC-TP), in retinal degeneration protein, type B (RdgB)-related PI-TPs (e.g. Dm RdgB, Nir2, Nir3) and in peroxisomal beta-oxidation enzyme-related SCP-2 (i.e. 3-oxoacyl-CoA thiolase, also denoted as SCP-X and the 80-kDa D-bifunctional protein). Further I will summarize the most recent studies pertaining to the physiological function of these soluble phospholipid transfer proteins in metazoa.

Studies on multi-group multi-criteria decision-making problems for oil spill contingency management are in their infancy. This paper presents a second-order fuzzy comprehensive evaluation (FCE) model to resolve decision-making problems in the area of contingency management after environmental disasters such as oil spills. To assess the performance of different oil combat strategies, second-order FCE allows for the utilization of lexical information, the consideration of ecological and socio-economic criteria and the involvement of a variety of stakeholders. On the other hand, the new approach can be validated by using internal and external checks, which refer to sensitivity tests regarding its internal setups and comparisons with other methods, respectively. Through a case study, the Pallas oil spill in the German Bight in 1998, it is demonstrated that this approach can help decision makers who search for an optimal strategy in multi-thread contingency problems and has a wider application potential in the field of integrated coastal zone management.

Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein alpha (PI-TPalpha) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI-TPalpha are known to produce and secrete bioactive survival factors that protect fibroblasts against UV-induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI-TPalpha (CMalpha) has neuroprotective effects on primary neurons in culture. We show that CMalpha is capable of protecting primary, spinal cord-derived motor neurons from serum deprivation-induced cell death. Since the conditioned medium of wild-type cells was much less effective, we infer that the neuroprotective effect of CMalpha is linked (in part) to the PI-TPalpha-dependent production of arachidonic acid metabolites. The neuroprotective activity of CMalpha is partly inhibited by suramin, a broad-spectrum antagonist of G-protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI-TPalpha, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI-TPalpha could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders.

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed ("dark") accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.

Photodynamic treatment (PDT) is an emerging procedure for the therapy of cancer, based on photosensitizers, compounds that generate highly reactive oxygen species on illumination with visible light. Photodynamic peroxidation of cellular lipids is a consequence of PDT associated with cytolethality. We used chloromethyl dichlorodihydrofluorescein diacetate and a novel fluorescent ratiometric oxidation-sensitive probe, C11-BODIPY581/591 (C11-BO), which reports on lipid peroxidation, for visualizing oxidative stress in cells subjected to PDT with a phthalocyanine photosensitizer Pc4. With C11-BO loaded into the cells before or immediately after PDT, we observed a prolonged oxidation, which continued up to 30 min after illumination. In contrast, H2O2 caused oxidation of C11-BO only when the cells were in direct contact with H2O2. PDT-induced oxidative stress was most pronounced in vesicular perinuclear organelles, most likely photodamaged lysosomes. We hypothesize that the lysosomal localization of the prolonged oxidative stress is a consequence of the presence of redox-active iron in lysosomes. In conclusion, we have found that oxidative stress induced in cells by PDT differs from one induced by H2O2 in respect of induction of prolonged oxidation of lipids.

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.

Mouse fibroblast cells overexpressing phosphatidylinositol transfer protein alpha [PI-TPalpha; sense PI-TPalpha (SPIalpha) cells] show a significantly increased rate of proliferation and an extreme resistance toward ultraviolet- or tumor necrosis factor-alpha-induced apoptosis. The conditioned medium (CM) from SPIalpha cells or the neutral lipid extract from CM stimulated the proliferation of quiescent wild-type NIH3T3 cells. CM was also highly effective in increasing resistance toward induced apoptosis in both wild-type cells and the highly apoptosis-sensitive SPIbeta cells (i.e., wild-type cells overexpressing PI-TPbeta). CM from SPIalpha cells grown in the presence of NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, expressed a diminished mitogenic and antiapoptotic activity. This strongly suggests that at least one of the bioactive factor(s) is an eicosanoid. In accordance, SPIalpha cells express enhanced levels of COX-1 and COX-2. The antiapoptotic activity of CM from SPIalpha cells tested on SPIbeta cells was inhibited by approximately 50% by pertussis toxin and suramin as well as by SR141716A, a specific antagonist of the cannabinoid 1 receptor. These inhibitors had virtually no effect on the COX-2-independent antiapoptotic activity of CM from SPIalpha cells. The latter results imply that PI-TPalpha mediates the production of a COX-2-dependent eicosanoid that activates a G-protein-coupled receptor, most probably a cannabinoid 1-like receptor., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10-35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H(2)O(2)-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular "ATP-meter(s)" generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).

Phosphatidylinositol transfer proteins (PI-TPs) consist of two isoforms (PI-TPalpha and PI-TPbeta), which differ in phospholipid transfer properties and intracellular localization. Both PI-TP isoforms are substrates for protein kinase C and contain a minor phosphorylation site (Ser166 in PI-TPalpha; Ser165 in PI-TPbeta). Only PI-TPbeta contains a major phosphorylation site at Ser262, which must be phosphorylated for PI-TPbeta to be associated with the Golgi. The PI-TP isoforms are completely conserved between mammals. Although their function is still not clear, their importance follows from knock-out studies, showing that mice lacking PI-TPalpha die soon after birth and that embryonic stems cells lacking PI-TPbeta cannot be generated [Mol. Biol. Cell 13 (2002) 739]. We determined the levels of the PI-TP isoforms in various mouse tissues by immunoblotting. PI-TPalpha is present in all tissues investigated, with highest levels in brain (167 ng/100 microg total protein). The levels of PI-TPbeta are 50-100 times lower than those of PI-TPalpha, with relatively high levels found in liver and brain (1.2 and 1.8 ng/100 microg of total protein, respectively). In contrast to NIH3T3 cells overexpressing PI-TPalpha, cells overexpressing PI-TPbeta (SPIbeta cells) were able to maintain steady-state levels of sphingomyelin in plasma membrane under conditions where this lipid is degraded by exogenous sphingomyelinase. This process of rapid sphingomyelin replenishment is dependent on PI-TPbeta being associated with the Golgi as cells overexpressing a mutant PI-TPbeta in which the major phosphorylation site is replaced (PI-TPbeta(S262A) behave as wild-type NIH3T3 cells. Since the SPIbeta cells display a decreased growth rate (35 h as compared to 21 h for wtNIH3T3 cells), we have investigated the sensitivity of these cells towards UV-induced apoptosis. We have found that the SPIbeta cells, but not the cells overexpressing PI-TPbeta(S262A), are very sensitive. We are currently investigating whether a relationship exists between PI-TPbeta being involved in maintaining plasma membrane sphingomyelin levels and the enhanced sensitivity towards apoptosis.

The SCP gene encodes two proteins, sterol carrier protein X (SCPx) and SCP2, that are independently regulated by separate promoters. SCPx has been shown to be the thiolase involved in the breakdown of branched-chain fatty acids and in the biosynthesis of bile acids. The in vivo function of SCP2 however remains to be established. The transcriptional regulation of SCPx and SCP2 is unclear, but their promoter regions contain several putative regulatory domains. We show here that both SCPx and SCP2 are upregulated by the daf-16-like Forkhead transcription factor FOXO3a (also known as FKHRL1) on the level of promoter activity. It was recently described that Forkheads regulate protection against (oxidative) stress in both Caenorhabditis elegans and mammalian cells. We looked into a role for SCP2 in the cellular defense against oxidative damage and found that a fluorescent fatty acid analog bound to SCP2 is protected against H2O2/Cu2+-induced oxidative damage. We propose a model for the way in which SCP2 could protect fatty acids from peroxidation.

An integrated modeling framework was developed to assess physical, biological and chemical processes in the sediment and at the sediment-water interface. Special focus is laid on the description of different functional groups of bacteria as defined according to their metabolic pathways, including fermentation, methanogenesis and oxidation of high and low molecular mass dissolved organic carbon, ammonium as well as other reduced compounds. The model is subjected to a new validation method which allows for an appropriate representation of remaining uncertainties. It is also able to reproduce two-dimensional gradients in all state variables induced by a pore-water velocity field typical for permeable sediments. Another improvement with respect to many classical models follows from the simulation of adaptive changes in dormancy and motility strategies. Within an extensive analysis stage, the evolutionary stability of these strategies is investigated under a variable hydrodynamical regime. The results show that optimal behavior in terms of adhesion and readiness to dormancy shifts differ between functional groups. This pattern is compared to recent empirical findings and discussed in relation to the confidence limits of the overall methodology. In the numerical experiments, also the effect of variable microbial strategies on the total carbon mineralization of the sediment is determined.

Photodynamic treatment (PDT) is an emerging therapeutic procedure for the management of cancer, based on the use of photosensitizers, compounds that generate highly reactive oxygen species (ROS) on irradiation with visible light. The ROS generated may oxidize a variety of biomolecules within the cell, loaded with a photosensitizer. The high reactivity of these ROS restricts their radius of action to 5-20 nm from the site of their generation. We studied oxidation of intracellular proteins during PDT using the ROS-sensitive probe acetyl-tyramine-fluorescein (acetylTyr-Fluo). This probe labels cellular proteins, which become oxidized at tyrosine residues under the conditions of oxidative stress in a reaction similar to dityrosine formation. The fluorescein-labeled proteins can be visualized after gel electrophoresis and subsequent Western blotting using the antibody against fluorescein. We found that PDT of rat or human fibroblasts, loaded with the photosensitizer Hypocrellin A, resulted in labeling of a set of intracellular proteins that was different from that observed on treatment of the cells with H2O2. This difference in labeling patterns was confirmed by 2D electrophoresis, showing that a limited, yet distinctly different, set of proteins is oxidized under either condition of oxidative stress. By matching the Western blot with the silver-stained protein map, we infer that alpha-tubulin and beta-tubulin are targets of PDT-induced protein oxidation. H2O2 treatment resulted in labeling of endoplasmic reticulum proteins. Under conditions in which the extent of protein oxidation was comparable, PDT caused massive apoptosis, whereas H2O2 treatment had no effect on cell survival. This suggests that the oxidative stress generated by PDT with Hypocrellin A activates apoptotic pathways, which are insensitive to H2O2 treatment. We hypothesize that the pattern of protein oxidation observed with Hypocrellin A reflects the intracellular localization of the photosensitizer. The application of acetylTyr-Fluo may be useful for characterizing protein targets of oxidation by PDT with various photosensitizers.

Previous work showed that from all cellular proteins, the endoplasmic reticulum (ER) resident proteins are most sensitive to oxidative stress [hydrogen peroxide (H(2)O(2))], as determined using the oxidation-sensitive, membrane-permeable, acetylTyrFluo probe. Because of the importance of these proteins in proper cellular functioning, we studied (a) whether modifying the cellular redox state/antioxidant status alters the susceptibility of those proteins toward H(2)O(2) oxidative stress and (b) whether H(2)O(2) affects ER function with regard to protein folding. The cellular redox and/or antioxidative capacity was modified in several ways. Lowering the capacity increased H(2)O(2)-induced protein oxidation, and increasing the capacity lowered H(2)O(2)-induced protein oxidation. The effect of H(2)O(2) on ER-related protein maturation was investigated, using the maturation of the low-density lipoprotein receptor as a model. Its maturation was not affected at low concentrations of H(2)O(2) (< or = 400 micro M), which do result in oxidation of ER resident proteins. Maturation was slowed down or reversibly inhibited at higher concentrations of H(2)O(2) (1.5-2.0 mM). These results might be caused by several events, including oxidation of the low-density lipoprotein receptor itself or ER resident proteins resulting in decreased folding (capacity). Alternatively, oxidation of cytosolic proteins involved in ER Golgi transport might attenuate transport and maturation. Clearly, the mechanism(s) responsible for the impairment of maturation need further investigation.

Carbon allocation and regulation of specific leaf area (sigma) define key processes underlying the adaptation of plants to varying habitats. In this study, the general principles governing adaptation and a dynamic optimality model of plant adaptation are reviewed. The central new elements of this model are: (i) differential root carbon costs for maintaining a defined nutrient status; (ii) a simple formula for optimal sigma at steady-state as a function of nitrogen (N) status and irradiance; and (iii) generic rules for the time propagation of adapting traits. The model was applied to a large data set compiled by Ingestad et al. (1995) and McDonald et al. (1986a, 1986b) for birch seedlings (Betula pendula Roth) during stationary logarithmic growth and during transient changes in response to a range of irradiances and nutrient supply rates. In the stationary case, large variations in the fraction of leaf dry mass to total dry mass (f(L)), sigma and N concentration were simulated with high accuracy. The independently calibrated model described the temporal response of seedlings following a sharp decrease in N supply, which includes phenomena such as the temporary C accumulation in leaves and damped oscillations in N concentration. Dynamics in sigma were more sensitive to variation in light than in N supply. Nevertheless, adaptive adjustments in f(L), sigma and N concentration were strongly coupled, underlining the relevance of a whole-plant perspective when modeling plant growth and regulation. The high coincidence between model calculations and measured values supports the notion that plant acclimation can be both understood and predicted as a growth-optimizing mechanism.

Elucidation of the three-dimensional structure of phosphatidylinositol transfer protein alpha (PI-TPalpha) void of phospholipid revealed a site of membrane association connected to a channel for phospholipid binding. Near the top of the channel specific binding sites for the phosphorylcholine and phosphorylinositol head groups were identified. The structure of this open form suggests a mechanism by which PI-TPalpha preferentially binds PI from a membrane interface. Modeling predicts that upon association of PI-TPalpha with the membrane the inositol moiety of bound PI is accessible from the medium. Upon release from the membrane PI-TPalpha adopts a closed structure with the phospholipid bound fully encapsulated. This structure provides new insights as to how PI-TPalpha may play a role in PI metabolism.

Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and we conclude that this model also extends to mammalian systems.

The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783-7788]. By exposing the cells to H(2)O(2) (0.1 mM for 10 min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [(35)S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the (35)S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the (35)S-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed co-localization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum.

Acclimation patterns in kinetic coefficients clearly demonstrate the limits of Monod's theory for the mathematical description of microbial growth. Focusing on E. coli grown under variable glucose levels, these patterns turn out to be highly diverse and sometimes even contradictory. Here, a new model based on an optimisation assumption is applied to a spectrum of adaptation phenomena, which are observed at steady-state as well as during transient situations. On the level of apparent kinetic coefficients, rates of adaptation are calculated depending on differential growth benefits. The resulting dynamics is bounded since maximum growth rate and substrate affinity are related by a non-linear trade-off. Long-term effects of phenotypic and genotypic changes under glucose limitation are robustly predicted by the model and explained in terms of their adaptive significance. Equivocal short-term recovery patterns occurring after sudden substrate excess are traced back to differences in the internal physiological state of the cells which in turn can be calculated in dependence on the inoculum history. Metabolic stress is a second determinant of short-term variations in kinetic coefficients which is here quantified in relation to external conditions as well as the internal state of cells. We demonstrate that lag phenomena and oscillations in anabolic activity exercised by E. coli under continuous growth acceleration can be reproduced without formulations being explicit in lag periods, metabolite concentrations or the timing of experimental changes. The overall predictive power of the simple approach indicates that slow as well as fast adjustments in apparent kinetic characteristics are strongly related to a dynamic optimisation strategy.

Protein oxidation of human umbilical-vein endothelial cells (HUVEC) in culture was examined under various conditions of oxidative stress. Extracellular protein (ECP) oxidation was assessed by determining dityrosine bond formation, which is indicated by the covalent coupling of the membrane-impermeable tyramine-fluorescein conjugate (TyrFluo) to HUVEC proteins. The acetylated membrane-permeable form of TyrFluo (acetylTyrFluo) was used for the determination of intracellular protein (ICP) oxidation. Oxidative stress was induced by exposing the HUVEC to PMA-activated human neutrophils, to a horseradish peroxidase/hydrogen peroxide (HRP/H(2)O(2)) system or to H(2)O(2) alone. Coupling of the probes was determined by confocal laser scanning microscopy and by Western blotting using anti-fluorescein antibody. Diethylamine nitric oxide (DEANO) was used to determine the effect of NO on the tyrosyl radical formation in proteins. The oxidative burst generated by activated neutrophils for 15 min, resulted in inducing dityrosine formation in ECP of HUVEC. Similar results were obtained with HRP/H(2)O(2), but H(2)O(2) alone did not have any effect on ECP. In the presence of DEANO (0.1 mM or higher), ECP oxidation was almost completely inhibited. This indicates that NO may protect endothelial cells against protein oxidation by activated neutrophils under pro-inflammatory conditions. Activated neutrophils did not oxidize ICP of HUVEC, which strongly suggests that the effect of the oxidative burst was restricted to the proteins exposed to the medium.

Phosphatidylcholine transfer protein (PC-TP) containing different molecular species of PC and phosphatidylinositol transfer protein alpha (PI-TPalpha) containing either a PI, PC, or PG molecule were identified as intact complexes by nano-electrospray ionization time-of-flight mass spectrometry. The stability of these complexes in the gas phase was determined by elevating the cone voltage (cv) resulting in the appearance of the protein void of lipid. PC-TP containing a PC species carrying an sn-1 palmitoyl chain was less stable than PC-TP containing a PC species carrying an sn-1 stearoyl chain given that these complexes were dissociated for 50% at a cv of roughly 30 and 45 V, respectively. Different acyl chains on the sn-2 position did not lead to significant changes in stability of the complex. In the case of PI-TPalpha, the complexes containing PI and PG were dissociated for 50% at a cv of 100 V as compared to a cv of 40 V for the complex containing PC. We propose that this difference in stability is due to hydrogen bonds between the polar headgroup of PI and PG and the lipid-binding site of PI-TPalpha. This may explain why PI-TPalpha preferentially binds PI from a membrane interface.

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

Phosphatidylinositol transfer protein alpha (PITP alpha) is a ubiquitous and highly conserved protein in multicellular eukaryotes that catalyzes the exchange of phospholipids between membranes in vitro and participates in cellular phospholipid metabolism, signal transduction and vesicular trafficking in vivo. Here we report the three-dimensional crystal structure of a phospholipid-free mouse PITP alpha at 2.0 A resolution. The structure reveals an open conformation characterized by a channel running through the protein. The channel is created by opening the phospholipid-binding cavity on one side by displacement of the C-terminal region and a hydrophobic lipid exchange loop, and on the other side by flattening of the central beta-sheet. The relaxed conformation is stabilized at the proposed membrane association site by hydrophobic interactions with a crystallographically related molecule, creating an intimate dimer. The observed open conformer is consistent with a membrane-bound state of PITP and suggests a mechanism for membrane anchoring and the presentation of phosphatidylinositol to kinases and phospholipases after its extraction from the membrane. Coordinates have been deposited in the Protein Data Bank (accession No. 1KCM).