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Our work is motivated by the empirical finding that Benefit corporations (B corps) that pursue certification experience a short-term slowdown in financial performance. To shed light on this finding...

Introduction Inherited mitochondrial DNA (mtDNA) variants may influence Alzheimer's disease (AD) risk. Methods We sequenced mtDNA from 146 AD and 265 cognitively normal (CN) subjects from the University of Kansas AD Center (KUADC) and assigned haplogroups. We further considered 244 AD and 242 CN AD Neuroimaging Initiative (ADNI) subjects with equivalent data. Results Without applying multiple comparisons corrections, KUADC haplogroup J AD and CN frequencies were 16.4% versus 7.6% (P = .007), and haplogroup K AD and CN frequencies were 4.8% versus 10.2% (P = .063). ADNI haplogroup J AD and CN frequencies were 10.7% versus 7.0% (P = .20), and haplogroup K frequencies were 4.9% versus 8.7% (P = .11). For the combined 390 AD and 507 CN cases haplogroup J frequencies were 12.8% versus 7.3% (P = .006), odds ratio (OR) = 1.87, and haplogroup K frequencies were 4.9% versus 9.5% (P = .010), OR = 0.49. Associations remained significant after adjusting for apolipoprotein E, age, and sex. Conclusion This exploratory analysis suggests inherited mtDNA variants influence AD risk.

This paper investigates the outcomes of a policy experiment, the NSF SBIR/IUCRC Membership Supplement, designed to promote the success of small high-tech entrepreneurial ventures by providing subsidized memberships in university-based cooperative research centers (IUCRCs). Data collected via semi-structured interviews with representatives of 61 Small Business Innovation Research (SBIR) firms indicated that SBIR firms who used the supplement to join an IUCRC reported multiple R&D benefits including research cost avoidance, research savings, and access to expensive equipment. A vast majority of SBIR firms also reported realizing or anticipated realizing commercial benefits (e.g., new investors, new products, and improvements to existing products). As suggested by social capital theory, SBIR firms reported that the policy mix experiment helped them make new connections with faculty and industry. Following our qualitative results, a structural equation model was applied to test the effect of social capital as an antecedent of SBIR firm R&D and commercialization outcomes. Results suggested that the SBIR firms who developed more social capital through interactions with faculty and industry members realized significantly more R&D and commercialization benefits. Further, commercialization benefits mediated the relationship between social capital and the SBIR firm’s perceived return on investment. Overall, this study demonstrates the feasibility of subjecting mixed policy interventions to evaluative scrutiny and provides evidence that such instruments can have substantive and positive effects on small high-tech entrepreneurial ventures. We discuss implications for social capital theory, policy mix initiatives and entrepreneurship policy.

Overview: Digital transformation is reshaping entire segments and industries: communications, retail, and, increasingly, health care, medicine, agriculture, and manufacturing. While a few companies...

Hintergrund Das mobile (m) Health-Telemonitoring beschreibt eine Form der Telemedizin, bei der Smartphones, entsprechende mobile Apps und Sensoren zur Fernüberwachung von PatientInnen genutzt werden. Diese Technologie könnte besonders bei BewohnerInnen von Alters- und Pflegeeinrichtungen vorteilhaft sein. Aufgrund von Alter, Grunderkrankungen und eingeschränkter Mobilität sind diese BewohnerInnen gegenüber pulmonalen Infekten, inkl. COVID-19, besonders empfindlich. Ziel Entwicklung eines Konzepts zur Implementierung von mHealth Telemonitoring des kardiopulmonalen Systems bei BewohnerInnen von Alters- und Pflegeeinrichtungen. Studie Bei den BewohnerInnen werden über einen Zeitraum von 2 bis 12 Wochen mit den zur Verfügung gestellten digitalen Sensoren (Pulsoxymeter, Blutdruckmessgerät, Spirometer, Aktivitätstracker) tägliche Messungen durchgeführt. Aufgrund der erwarteten geringen digitalen Kenntnis der BewohnerInnen, wird das Pflegepersonal in die Messungen mit involviert und geschult. Bei Bedarf werden den BewohnerInnen Smartphones auf Leihbasis (eingelegt über Scheinaccounts) zur Verfügung gestellt. Die Akzeptanz des mHealth-Telemonitorings wird bei BewohnerInnen, Pflegepersonal und zuständigen Allgemein- bzw Fachärzten über eine Mixed-Methods Studie evaluiert, angelehnt an das Technologieakzeptanzmodell. Erwartete Ergebnisse und Schlussfolgerung Aufgrund der mangelnden digitalen Kenntnisse können ProbandInnen Probleme im Umgang mit den digitalen Sensoren haben. Der geistige bzw. körperliche Gesundheitszustand kann eine Herausforderung darstellen. Die Pflegekräfte könnten aufgrund Arbeitsbelastung nicht in der Lage sein, eine ausreichende Unterstützung zu leisten. Das betreuende Medizinpersonal könnte es schwierig finden, die Telemedizin mit ihrer üblichen Versorgung zu verbinden, nicht zuletzt wegen eines aktuell unklaren Kostenerstattungsmodells. Durch Beobachtung und Lösung der Hürden, sowie durch die Mixed-Methods Untersuchung, wird ein Konzept zur Implementierung vom mHealth Telemonitoring des kardiopulmonalen Systems in Alters- und Pflegeeinrichtungen vorangebracht.

We report on the findings from an in-class experiment that represents a learning innovation which can enable classroom-based conversations about bias in the domain of entrepreneurship. More specifically, the present learning innovation explores gender bias in venture funding with regard to entrepreneurship. In an introduction to entrepreneurship class, we randomly assigned students to one of the three experimental conditions—students evaluated an executive summary for a venture either written by a woman, or a man, or one in which the gender was neutral (i.e., the control group). Students acted as if they were considering an investment and reported whether, for example, the executive summary was well written as well as how much equity they would want in the venture as a potential investor. Overall, these results provide evidence consistent with the inference that the students sampled in this study did not use gender as a decision-making heuristic when evaluating entrepreneurial opportunities. We discuss the results of our experiment and describe (a) how to replicate this activity, (b) how to discuss this in the classroom, and (c) how to adapt this activity to explore other types of bias (e.g., race, ethnicity, weight-based, etc.).

The act of being resourceful is a commonly displayed behavior in the process of entrepreneurship. For example, entrepreneurs are known to share resources with competitors, utilize their social networks to attract capital, exchange favors for resources, engage in resource bootstrapping behaviors, repurpose and/or recombine existing resources for new purposes (i.e., bricolage), and sometimes pivot from one opportunity to another following available resource options given current situational constraints (i.e., effectuation). Currently, research on the topic of resourcefulness in the entrepreneurship literature assumes these aforementioned resourceful behaviors are attributed to a limited resource environment rather than also originating from within the entrepreneur. Frugality is a new concept in the field of entrepreneurship that suggests entrepreneurs will also enact resourceful behaviors because of their own self-regulatory processes; that is, entrepreneurs will engage in resourcefulness behaviors as a preference rather than as a forced reaction to their external resource environment. Thus, frugality represents an individual-level antecedent of resourcefulness behaviors that is not bound to the conditions of necessity-based entrepreneurship. This is important as frugality opens the door for numerous future research directions in the context of both necessity-based and opportunity-based entrepreneurship. Frugality is defined as one’s general preference to (a) conserve resources and (b) apply an economic rationale in the acquisition of resources (i.e., assessing the opportunity cost of newly acquired resources). Research in the consumer behavior literature highlights that frugality is a culturally driven trait preference, whereby one is willing to sacrifice in the short term to achieve longer-term, idiosyncratic goals. Despite a large amount of research on frugal consumer behavior, there has yet to be a systematic inquiry into how frugality more broadly influences the process of new venture creation and organizations. Empirical research highlights that frugal entrepreneurs tend to engage in higher amounts of bricolage and effectuation, thus representing a promising new topic for better understanding the process of entrepreneurship. Although it is expected that future inquiry regarding frugality in entrepreneurship will naturally orient toward the topic of resourcefulness, it is also expected that frugality will relate to numerous other important topics such as entrepreneurial well-being, opportunity recognition, opportunity exploitation, and new venture growth. Considering the novelty of frugality in entrepreneurship, and management literature generally, it would benefit future research to systematically explore both the upsides and downsides to being frugal as it relates to value creation activities.

What should researchers say when recruiting entrepreneurs to participate in their study? Using a sample of entrepreneurs (N = 1,450) who were being asked to participate in an academic research project, we conducted an experiment to determine recruitment message efficacy. Drawing on best practices from the behavioral insights literature, we developed different email message recruitment statements that were randomly assigned across four phases of our experiment. Results indicate that a message grounded in the “descriptive norms” (i.e., social norms) approach resulted in the highest percentage of participants who clicked on the link to participate in our online survey. We discuss the theoretical as well as practical implications of our work.

We integrate social cognitive theory, and its tenets of personal and collective agency, to develop an individual-level perspective on entrepreneurs' resourcefulness behaviors that illustrates how resourcefulness behaviors can be classified as ‘self-reliant behaviors’ or ‘joint resourcefulness behaviors’. Using this novel cognitive theoretical approach, we provide and test a framework that explains how dispositional, perceptual, and behavioral factors interact in the enactment of purposeful action with regards to entrepreneurs' resourceful behaviors. Consistent with our hypotheses, results from a quantitative study of entrepreneurs (N = 178), as well as a supplemental study involving qualitative interviews with entrepreneurs (N = 15), highlight that entrepreneurs higher in frugality tend to perceive higher levels of environmental hostility. This relationship, in turn, leads to higher amounts of self-reliant resourcefulness behaviors (i.e., customer-related and internal self-financing bootstrapping behaviors) but not joint resourcefulness behaviors. Multiple theoretical and practical contributions emerge from our findings as the extant literature does not yet account for human agency as a reason why some entrepreneurs may choose to engage in certain resourceful behaviors relative to other behaviors.

We examine how innovation culture affects new product launch performance in a sample of entrepreneurial ventures (N = 334). In order to examine the relation between innovation culture and new product launch performance, we embarked on a two-step process. First, we examined the factor structure of innovation culture—results confirmed nine-dimensions. Here, a grouped confirmatory factor analysis (CFA) examined cross-cultural differences between eastern and western cultures. Second, the dimensions of innovation culture were used to determine culture profiles across these 334 entrepreneurial ventures. We found two clear subpopulations of innovation culture and we link these two clusters to new product development (NPD) performance (i.e., new product sales and profits averaged over 5 years). In particular, a Latent Profile Analysis (LPA) suggested two profiles existed with respect to innovation culture—that is, ventures that ranked ‘high’ across all innovation culture dimensions versus ventures that ranked ‘low’ across all nine dimensions. Ventures scoring higher across all innovation culture dimensions had significantly higher new product profits and sales. In a series of robustness checks, a path model revealed no significant moderation by region (i.e., eastern vs. western countries) in the innovation culture to performance relationship. Implications as well as directions for future research are discussed.

This study tests a situated metacognitive model of entrepreneurial action to highlight how action (or inaction) during the entrepreneurial process is influenced by both individual traits and one's metacognitive ability, namely one's strategic mindset. Integrating theory on resourcefulness and metacognition, we show how entrepreneurs who are more frugal tend to engage in less action in developing their new venture (i.e., enacting fewer innovative behaviors and putting forth less effort) as compared to less frugal entrepreneurs. However, we explain that this direct (negative) relationship is mediated by one's strategic mindset, such that the indirect effect of frugality on both innovative behavior and level of effort enacted towards one's new venture is positive (rather than negative). Overall, this study extends the construct of strategic mindset to the entrepreneurship literature and highlights the crucial role that metacognition can play regarding one's socio-cognitive decision-making process and subsequent entrepreneurial behaviors.

This article describes the results of a survey designed to assess how companies are implementing digital transformation, including the various strategies they employ and the actions they take to achieve large-scale transformations. While a few companies seem to reach front-runner status, the majority seem to lag behind. This phenomenon is a top concern of boardrooms worldwide and motivated the development of this study. To help these organizations, we highlight differentiated strategic principles and characteristics of the companies’ design processes digitally mature companies undertake to transform their businesses. These insights should help lagging companies understand what is involved in implementing a digital transformation and what they need to do to enforce this transformation.

The present research complements extant perspectives of resourcefulness, which assert that resourceful behaviors arise out of responses to environmental constraints, by developing a model illustrating that entrepreneurs self-impose constraints on resource acquisition and deployment for differing reasons. Specifically, we introduce a novel conceptualization of frugality and differentiate it from self-control to develop a set of hypotheses that frugality predicts resource use behaviors based on long-held preferences (e.g., effectuation and bricolage) and self-control predicts resource use behaviors based on known end states or goals (e.g., causation and pre-commitments). After accumulating evidence of reliability and validity for a new measure of frugality contextualized for entrepreneurship research, the results support our self-regulatory theoretical framework. Our study contributes to research on resourcefulness by making multiple theoretical insights, and we outline numerous future research opportunities for applying the construct of frugality to explain entrepreneurial behavior.

Control of intracellular calcium concentrations ([Ca 2 + ] i ) is essential for neuronal function, and the plasma membrane Ca 2 + -ATPase (PMCA) is crucial for the maintenance of low [Ca 2 + ] i . We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca 2 + homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d -threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( d -PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d -PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca 2 + transporter.

Hybrid entrepreneurship—where an individual simultaneously engages in startup activities as well as wage-based employment—is an increasingly common career transition path. Yet, relatively little research has explored entrepreneurial characteristics during these unique career transitions. We provide exploratory insight into the longitudinal relationship between self-efficacy and persistence for hybrid entrepreneurs’ (N = 29) across a twenty-week period during which aspiring entrepreneurs engaged in activities related to venture startup while maintaining wage employment. As such, we propose that entrepreneurial self-efficacy (ESE) and entrepreneurial persistence are malleable concepts that change over time. Using our longitudinal sample, we find evidence that ESE predicts entrepreneurial persistence change over time. Perhaps more importantly, we model how changes in ESE over time affect changes in entrepreneurial persistence for hybrid entrepreneurs. In sum, our work provides a foundation from which future research can examine the longitudinal transition of nascent entrepreneurs moving from an occupational setting as an employee to launching their own venture.

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

Context: Individual patient prognostication for advanced thyroid cancer (TC) is challenging. Circulating tumor cells (CTCs) have been shown to be a valuable prognostic marker for other solid cancers. Objective: We hypothesized that CTCs are present in the blood of patients with advanced TC and their number can predict overall survival (OS). Setting: This is a prospective study at a tertiary cancer hospital. Patients, Interventions, and Main Outcome Measures: Initial studies were performed with TC cell lines to determine the feasibility of detection using the Veridex CellSearch. CTC enumeration was performed in blood samples from 18 patients with distantly metastatic medullary TC (metMTC), 14 with distantly metastatic differentiated TC (metDTC), and 10 controls with a history of TC but no evidence of disease. The prognostic value of CTC levels to predict OS in metMTC patients was assessed. Results: CellSearch detected cells from MTC and DTC but not anaplastic TC cell lines. Six metMTC patients but no metDTC or control patients had more than or equal to 5 CTCs detected by the CellSearch assay. Median survival in metMTC patients with more than or equal to 5 CTCs was 13 months vs 51.5 months for those with less than 5 CTCs (P = .0116). The hazard ratio for mortality of patients with more than or equal to 5 CTCs compared with those with less than 5 CTCs was 3.95 (1.20–13.0, P = .0245). Conclusions: The presence of more than or equal to 5 CTCs in patients with metMTC is associated with worse OS. Larger cohorts are required to validate the prognostic value of CTC enumeration.

Precise regulation of free intracellular Ca2+ concentrations [Ca2+]i is critical for normal neuronal function, and alterations in Ca2+ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca2+]i is the plasma membrane Ca2+-ATPase (PMCA), the high-affinity transporter that fine tunes the cytosolic nanomolar levels of Ca2+. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non-raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age-related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.

Impaired axonal transport has been linked to the pathogenic processes of Alzheimer’s disease (AD) in which axonal swelling and degeneration are prevalent. The development of non-invasive neuroimaging methods to quantitatively assess in vivo axonal transport deficits would be enormously valuable to visualize early, yet subtle, changes in the AD brain, to monitor the disease progression and to quantify the effect of drug intervention. A triple transgenic mouse model of AD closely resembles human AD neuropathology. In this study, we investigated age-dependent alterations in the axonal transport rate in a longitudinal assessment of the triple transgenic mouse olfactory system, using fast multi-sliced T1 mapping with manganese-enhanced MRI. The data show that impairment in axonal transport is a very early event in AD pathology in these mice, preceding both deposition of Aβ plaques and formation of Tau fibrils.

Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as ‘lipid rafts’ contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed age-dependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p < 0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain.

Pivotal brain functions, such as neurotransmission, cognition, and memory, decline with advancing age and, especially, in neurodegenerative conditions associated with aging, such as Alzheimer's disease (AD). Yet, deterioration in structure and function of the nervous system during aging or in AD is not uniform throughout the brain. Selective neuronal vulnerability (SNV) is a general but sometimes overlooked characteristic of brain aging and AD. There is little known at the molecular level to account for the phenomenon of SNV. Functional genomic analyses, through unbiased whole genome expression studies, could lead to new insights into a complex process such as SNV. Genomic data generated using both human brain tissue and brains from animal models of aging and AD were analyzed in this review. Convergent trends that have emerged from these data sets were considered in identifying possible molecular and cellular pathways involved in SNV. It appears that during normal brain aging and in AD, neurons vulnerable to injury or cell death are characterized by significant decreases in the expression of genes related to mitochondrial metabolism and energy production. In AD, vulnerable neurons also exhibit down-regulation of genes related to synaptic neurotransmission and vesicular transport, cytoskeletal structure and function, and neurotrophic factor activity. A prominent category of genes that are up-regulated in AD are those related to inflammatory response and some components of calcium signaling. These genomic differences between sensitive and resistant neurons can now be used to explore the molecular underpinnings of previously suggested mechanisms of cell injury in aging and AD.

A variety of approaches have been taken to improve the brain penetration of pharmaceutical agents. The amphipathic character of a compound can improve its interaction with the lipid bilayer within cell membranes, and as a result improve permeability. Fatty acid chains or lipoamino acids of various lengths were attached to tranylcypromine (TCP), in an attempt to improve the blood-brain barrier (BBB) permeability by increasing the lipophilicity as well as the amphiphatic character of the drug. TCP-FA4, one of the derivatives containing a four carbon alkyl acid chain, showed the greatest improvement in permeability. This molecule was slightly neuroprotective in a beta-amyloid-induced neurodegeneration assay and may also be capable of upregulating brain derived neurotrophic factor (BDNF), as indicated by cell culture assays using human umbilical vein endothelial cells. Since decreased levels of BDNF are observed in many CNS disorders, and direct injection of BDNF is not a viable option due to its poor permeability across the BBB, small molecules capable of regulating BDNF that also cross the BBB may be an interesting treatment option.

Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium ([Ca(2+)](i)) are not known. The calmodulin (CaM)-stimulated plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in regulating [Ca(2+)](i). Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would probably occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5-10 microM) increased PMCA basal activity by two-fold but abolished its sensitivity to CaM. Higher concentrations (25-100 microM) inhibited both components of PMCA activity. Immunoblots showed the formation of high-molecular-weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca(2+) transporter under prolonged oxidative stress could alter neuronal Ca(2+) signaling.

Compounds that interact with microtubules, such as paclitaxel, have been shown to possess protective properties against beta-amyloid (Abeta) induced neurodegeneration associated with Alzheimer's disease. In this work, the novel agent (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol was investigated for effectiveness in protecting neurons against several toxic stimuli and its interaction with the microtubule network. Exposure of neuronal cultures to Abeta peptide in the presence of 5 nM (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol resulted in a 50% increase in survival. Neuronal cultures treated with other toxic stimuli such as staurosporine, thapsigargin, paraquat, and H(2)O(2) showed significantly enhanced survival in the presence of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol. Microtubule binding and tubulin assembly studies revealed differences compared to paclitaxel but confirmed the interaction of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol with microtubules. Furthermore, in vitro studies using bovine brain microvessel endothelial cells experiments suggest that (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol can readily cross the blood-brain barrier in a passive manner.

Alzheimer's disease (AD) neuropathology is characterized by loss of synapses and neurons, neuritic plaques consisting of beta-amyloid (Abeta) peptides, and neurofibrillary tangles consisting of intracellular aggregates of hyperphosphorylated tau protein in susceptible brain regions. Abeta oligomers trigger a cascade of pathogenic events including tau hyperphosphorylation and aggregation, inflammatory reactions, and excitotoxicity that contribute to the progression of AD. The molecular chaperone Hsp90 facilitates the folding of newly synthesized and denatured proteins and is believed to play a role in neurodegenerative disorders in which the defining pathology results in misfolded proteins and the accumulation of protein aggregates. Some agents that inhibit Hsp90 protect neurons against Abeta toxicity and tau aggregation, and assays for rapidly screening potential Hsp90 inhibitors are of interest. We used the release of the soluble cytosolic enzyme lactate dehydrogenase (LDH) as an indicator of the loss of cell membrane integrity and cytotoxicity resulting from exposure to Abeta peptides to evaluate the neuroprotective properties of novel novobiocin analogues and established Hsp90 inhibitors. Compounds were assessed for potency in protecting proliferating and differentiated SH-SY5Y neuronal cells against Abeta-induced cell death; the potential toxicity of each agent alone was also determined. The data indicated that several of the compounds decreased Abeta toxicity even at low nanomolar concentrations and, unexpectedly, were more potent in protecting the undifferentiated cells against Abeta. The novobiocin analogues alone were not toxic even up to 10 microM concentrations whereas GDA and the parent compound, novobiocin, were toxic at 1 and 10 microM, respectively. The results suggest that novobiocin analogues may provide novel leads for the development of neuroprotective drugs.

Intraneuronal calcium ([Ca2+]i) regulation is altered in aging brain, possibly because of the changes in critical Ca2+ transporters. We previously reported that the levels of the plasma membrane Ca2+-ATPase (PMCA) and the Vmax for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNAi techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro– transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca2+]i revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca2+]i, slower recovery from KCl-induced Ca2+ transients, and incomplete return to pre-stimulation Ca2+ levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca2+ signaling and enhanced susceptibility to stresses that can alter the regulation of Ca2+ homeostasis.

Spatial and temporal alterations in intracellular calcium [Ca(2+)](i) play a pivotal role in a wide array of neuronal functions. Disruption in Ca(2+) homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca(2+)-ATPase (PMCA) is a high affinity Ca(2+) transporter that plays a crucial role in the termination of [Ca(2+)](i) signals and in the maintenance of low [Ca(2+)](i) essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca(2+) signaling in the central nervous system.

Ceramide is a bioactive sphingolipid that can prevent calpain activation and beta-amyloid (A beta) neurotoxicity in cortical neurons. Recent evidence supports A beta induction of a calpain-dependent cleavage of the cyclin-dependent kinase 5 (cdk5) regulatory protein p35 that contributes to tau hyperphosphorylation and neuronal death. Using cortical neurons isolated from wild-type and p35 knockout mice, we investigated whether ceramide required p35/cdk5 to protect against A beta-induced cell death and tau phosphorylation. Ceramide inhibited A beta-induced calpain activation and cdk5 activity in wild-type neurons and protected against neuronal death and tau hyperphosphorylation. Interestingly, A beta also increased cdk5 activity in p35-/- neurons, suggesting that the alternate cdk5 regulatory protein, p39, might mediate this effect. In p35 null neurons, ceramide blocked A beta-induced calpain activation but did not inhibit cdk5 activity or cell death. However, ceramide blocked tau hyperphosphorylation potentially via inhibition of glycogen synthase kinase-3beta. These data suggest that ceramide can regulate A beta cell toxicity in a p35/cdk5-dependent manner.

We tested how the addition of oxaloacetate (OAA) to SH-SY5Y cells affected bioenergetic fluxes and infrastructure, and compared the effects of OAA to malate, pyruvate, and glucose deprivation. OAA displayed pro-glycolysis and pro-respiration effects. OAA pro-glycolysis effects were not a consequence of decarboxylation to pyruvate because unlike OAA, pyruvate lowered the glycolysis flux. Malate did not alter glycolysis flux and reduced mitochondrial respiration. Glucose deprivation essentially eliminated glycolysis and increased mitochondrial respiration. OAA increased, while malate decreased, the cell NAD+/NADH ratio. Cytosolic malate dehydrogenase 1 protein increased with OAA treatment, but not with malate or glucose deprivation. Glucose deprivation increased protein levels of ATP citrate lyase, an enzyme which produces cytosolic OAA, whereas OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased cytochrome oxidase subunit 2, PGC1α, PGC1β, and PGC1 related co-activator protein levels. OAA increased total and phosphorylated SIRT1 protein. We conclude that adding OAA to SH-SY5Y cells can support or enhance both glycolysis and respiration fluxes. These effects appear to depend, at least partly, on OAA causing a shift in the cell redox balance to a more oxidized state, that it is not a glycolysis pathway intermediate, and possibly its ability to act in an anaplerotic fashion. We examined how oxaloacetate (OAA) affects bioenergetic fluxes. To advance the understanding of how OAA mediates these changes, we compared the effects of OAA to malate, pyruvate, and glucose deprivation. We further examined how OAA affects levels of enzymes that facilitate its cytosolic metabolism, and found OAA increased the expression of malate dehydrogenase 1 (MDH1-cytosolic). We propose the following: OAA supports both glycolysis and respiration fluxes, shifts the cell redox balance toward a more oxidized state, and acts in an anaplerotic fashion. Abbreviations not defined in the text: MDH2, malate dehydrogenase 2 (mitochondrial).

Most drug discovery efforts for Alzheimer's disease (AD) have focused on prevention or clearance of beta-amyloid (Abeta) fibrils or oligomers, with far less attention to prevention of tau abnormalities that lead to neurofibrillary tangles (NFTs). Much evidence now indicates that Abeta multimers can trigger neurodegenerative changes that involve formation of dystrophic neurites and cytoskeletal collapse, possibly due loss of microtubule (MT) stabilization by the tau protein. We have found that several MT-stabilizing agents such as Taxol significantly enhanced neuronal survival in the presence of Abeta and identified agents that enter the brain, a necessity for in vivo testing in animal models of tau pathology. Studies were designed to test two agents in the tau mutant (JNPL3) mouse that develops severe motor deficits at about seven months of age, accompanied by neuropathological markers of tau pathology. In addition to using motor performance tests through the planned period of drug administration, we designed a simple appetitive memory test that required a reduction in ad lib food intake. Although the neurochemical data are still being analyzed, we were surprised to find that all of the JNPL3 mice, whether receiving the drug or not, developed no signs of motor impairment up to 10 months of age. This is considerably beyond the age at which free-fed mice survived and suggests that the food restriction alone may have delayed the pathological process. A study is ongoing with free-fed mice to determine if the drug interventions do have any beneficial effects in these mutant mice.

We used single-molecule fluorescence spectroscopy to probe the conformation of calmodulin (CaM) bound to oxidatively modified plasma-membrane Ca(2+)-ATPase (PMCAox). We found that oxidative modification altered the coupling between the ATP binding domain and the autoinhibitory domain. Oxidative modification of PMCA is known to result in a loss of activity for the enzyme. Conformations of PMCAox-CaM complexes were probed by single-molecule polarization modulation spectroscopy, which measured the orientational mobility of fluorescently labeled CaM bound to PMCAox. We detected an enhanced population of PMCAox-CaM complexes with a low orientational mobility in the presence of ATP, whereas nonoxidized PMCA-CaM complexes existed almost exclusively in a high-mobility state in the presence of ATP. We have previously attributed such high-mobility states to PMCA-CaM complexes with a dissociated autoinhibitory/CaM binding domain, whereas the lower-mobility state was attributed to autoinhibited PMCA-CaM complexes with a nondissociated autoinhibitory domain [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. In the absence of ATP, the orientational mobility distributions are similar for CaM complexed with oxidized PMCA or nonoxidized PMCA. These results suggest that oxidative modification of PMCA reduced the propensity of the autoinhibitory domain to dissociate from binding sites near the catalytic core of the enzyme with bound nucleotide upon CaM stimulation in the presence of Ca(2+). This interpretation was further supported by chymotrypsin proteolysis, which probes the tightness of binding of the autoinhibitory domain to sites near the catalytic core of the enzyme. Enhanced proteolysis was observed for PMCA upon binding CaM or ATP. In contrast, proteolysis was partially blocked for oxidatively modified PMCA, even in the presence of ATP.

Settlement is almost always preferable to last-man-standing litigation. While litigation posturing is a necessity, all but the largest pharmaceutical houses must avoid suing to a conclusion. The objective is to make a reasonable deal. Having a non-lawyer corporate representative fully engaged in direct settlement efforts, with the assistance of counsel, throughout the process is one way to foster settlement. Also, be sure to propose a Scheduling Order with as many points of decision as possible. In particular, separate Markman claim construction briefings and rulings from summary judgment briefings and rulings. Preparing for the 95 per cent probability of settlement is prudent, cost effective and substantially less bloody. Abbreviated new drug application (ANDA) Paragraph IV litigation is a unique litigation exercise. Address ANDA Paragraph IV litigation and settlement separately in terms of how the ‘other side’ will react and how the Federal Trade Commission (FTC) will react. The objective is not to sustain or defeat a patent. These are merely aspects of potentially profitable constructions of a settlement agreement.