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Find video protocols related to scientific articles indexed in Pubmed.
Viral infection of engrafted human islets leads to diabetes.
Diabetes
PUBLISHED: 11-14-2014
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Type 1 diabetes (T1D) is characterized by the destruction of the insulin-producing ?-cells of pancreatic islets. Genetic and environmental factors both contribute to T1D development. Viral infection with enteroviruses is a suspected trigger for T1D but a causal role remains unproven and controversial. Studies in animals are problematic because of species-specific differences in host cell susceptibility and immune responses to candidate viral pathogens such as coxsackie B virus (CVB). In order to resolve the controversial role of viruses in human T1D, we developed a viral infection model in immunodeficient mice bearing human islet grafts. Hyperglycemia was induced in mice by specific ablation of native ?-cells. Human islets, which are naturally susceptible to CVB infection, were transplanted to restore normoglycemia. Transplanted mice were infected with CVB4 and monitored for hyperglycemia. Forty-seven percent of CVB4-infected mice developed hyperglycemia. Human islet grafts from infected mice contained viral RNA, expressed viral protein, and had reduced insulin compared to grafts from uninfected mice. Human-specific gene expression profiles in grafts from infected mice revealed the induction of multiple interferon stimulated genes. Thus, human islets can become severely dysfunctional with diminished insulin production following CVB infection of ?-cells, resulting in diabetes.
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Light-driven Na(+) pump from Gillisia limnaea: a high affinity Na(+) binding site is formed transiently in the photocycle.
Biochemistry
PUBLISHED: 11-07-2014
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A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper we examine a similar protein, GLR from Gillisia limnaea, expressed in E. coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations up to 3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+) - independent" to a "Na(+) - dependent" photocycle (or photocycle branches), which occurs at [Na(+)] of ca. 60 ?M. The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high affinity Na(+) binding site is created transiently after photoexcitation, and Na(+) entry from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counter ion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Na(+) binding to the mutant shifts the chromophore maximum to the red similarly to H(+), as occurs in the photocycle of the wild type.
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Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.
Science
PUBLISHED: 10-23-2014
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Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.
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Surveillance for brain metastases in patients receiving systemic therapy for advanced melanoma.
Melanoma Res.
PUBLISHED: 10-09-2014
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The objectives of this study were to determine the cumulative incidence and timing of new brain metastases over the course of systemic therapy for metastatic melanoma and to identify prognostic factors for brain metastases. Chemo-naive patients underwent computed tomography or MRI of the brain every 6 weeks. The cumulative incidence of confirmed brain metastases was calculated at 12-week intervals. Univariable and multivariable competing risk regression models were used to assess the association between the development of brain metastases and potential risk factors of interest. Cumulative incidence with competing risk and competing risk regression was used to assess the brain metastasis-free interval from the time of diagnosis of stage IV disease. The clinical characteristics of the 315 patients with brain metastases were compared with those of 370 brain metastasis-free patients. Among patients with brain metastases, a significantly higher proportion had stage M1b and M1c disease at diagnosis compared with stage M1a and a greater proportion had metastatic disease in three or more visceral sites. Significantly shorter brain metastasis-free intervals were found in these patients compared with patients with M1a disease and those with no visceral metastases. More than 80% of the 230 patients who developed brain metastases during systemic therapy had their brain metastases confirmed within 60 weeks from the onset of advanced melanoma. Imaging studies at 12-week intervals for 60 weeks after the diagnosis of advanced melanoma will detect brain metastases in most of the patients who will eventually develop them.
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The Effect of Polymeric Excipients on the Physical Properties and Performance of Amorphous Dispersions: Part I, Free Volume and Glass Transition.
Pharm. Res.
PUBLISHED: 08-09-2014
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To investigate the structural effect of polymeric excipients on the behavior of free volume of drug-polymer dispersions in relation to glass transition.
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TRIM13 is a negative regulator of MDA5-mediated type I interferon production.
J. Virol.
PUBLISHED: 07-09-2014
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Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13(-/-) mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13(-/-) mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13(-/-) murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(I · C) also show a significant increase in beta IFN (IFN-?) levels, but, in contrast, IFN-? responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection.
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Spectrum and mechanisms of inflammasome activation by chitosan.
J. Immunol.
PUBLISHED: 05-14-2014
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Chitosan, the deacetylated derivative of chitin, can be found in the cell wall of some fungi and is used in translational applications. We have shown that highly purified preparations of chitosan, but not chitin, activate the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in primed mouse bone marrow-derived macrophages (BMM?), inducing a robust IL-1? response. In this article, we further define specific cell types that are activated and delineate mechanisms of activation. BMM? differentiated to promote a classically activated (M1) phenotype released more IL-1? in response to chitosan than intermediate or alternatively activated macrophages (M2). Chitosan, but not chitin, induced a robust IL-1? response in mouse dendritic cells, peritoneal macrophages, and human PBMCs. Three mechanisms for NLRP3 inflammasome activation may contribute: K(+) efflux, reactive oxygen species, and lysosomal destabilization. The contributions of these mechanisms were tested using a K(+) efflux inhibitor, high extracellular potassium, a mitochondrial reactive oxygen species inhibitor, lysosomal acidification inhibitors, and a cathepsin B inhibitor. These studies revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, neither chitosan nor chitin stimulated significant release from unprimed BMM? of any of 22 cytokines and chemokines assayed. This study has the following conclusions: 1) chitosan, but not chitin, stimulates IL-1? release from multiple murine and human cell types; 2) multiple nonredundant mechanisms appear to participate in inflammasome activation by chitosan; and 3) chitin and chitosan are relatively weak stimulators of inflammatory mediators from unprimed BMM?. These data have implications for understanding the nature of the immune response to microbes and biomaterials that contain chitin and chitosan.
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Multidisciplinary management of advanced Basal cell carcinoma: report of four cases.
J Drugs Dermatol
PUBLISHED: 05-10-2014
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Advanced basal cell carcinomas (BCC) are neoplasms with high-risk clinical characteristics that can develop as locally advanced disease or metastasis. Treatment of advanced BCC may result in significant morbidity due to the technical challenges of size and/or location or in which surgery and radiation therapy may be contraindicated. No standard of care exists for the management of advanced BCC. As such, the difficulty in managing these tumors necessitates a multidisciplinary approach to patient care.
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Increased survival in B-cell-deficient mice during experimental cerebral malaria suggests a role for circulating immune complexes.
MBio
PUBLISHED: 03-20-2014
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The pathogenesis of malaria, an insect-borne disease that takes millions of lives every year, is still not fully understood. Complement receptor 1 (CR1) has been described as a receptor for Plasmodium falciparum, which causes cerebral malaria in humans. We investigated the role of CR1 in an experimental model of cerebral malaria. Transgenic mice expressing human CR1 (hCR1(+)) on erythrocytes were infected with Plasmodium berghei ANKA and developed cerebral malaria. No difference in survival was observed in hCR1(+) mice compared to wild-type mice following infection with P. berghei ANKA; however, hCR1 detection was significantly diminished on erythrocytes between days 7 and 10 postinfection. hCR1 levels returned to baseline by day 17 postinfection in surviving animals. Immunoblot assays revealed that total erythrocyte hCR1 levels were diminished, confirming that immune complexes in association with erythrocyte hCR1 were likely removed from erythrocytes in vivo by clearance following immune adherence. Decreases in hCR1 were completely dependent on C3 expression, as mice treated with cobra venom factor (which consumes and depletes C3) retained hCR1 on erythrocytes during C3 depletion through day 7; erythrocyte hCR1 decreases were observed only when C3 levels recovered on day 9. B-cell-deficient mice exhibit a marked increase in survival following infection with P. berghei ANKA, which suggests that immune complexes play a central role in the pathogenesis of experimental cerebral malaria. Together, our findings highlight the importance of complement and immune complexes in experimental cerebral malaria. IMPORTANCE Cerebral malaria is a deadly complication of infection with Plasmodium falciparum. Despite its high prevalence, relatively little is understood about its pathogenesis. We have determined that immune complexes are generated and deposited on erythrocytes specifically expressing human complement receptor 1 in a mouse model of cerebral malaria. We also provide evidence demonstrating the importance of immunoglobulins in the pathogenesis of cerebral malaria in mice. These findings may have important implications in human cerebral malaria.
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The metabolite ?-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.
Nature
PUBLISHED: 03-17-2014
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Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that ?-ketoglutarate (?-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ? is identified as a novel binding protein of ?-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that ?-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by ?-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by ?-KG requires ATP synthase subunit ? and is dependent on target of rapamycin (TOR) downstream. Endogenous ?-KG levels are increased on starvation and ?-KG does not extend the lifespan of dietary-restricted animals, indicating that ?-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.
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Dicer's role as an antiviral: still an enigma.
Curr. Opin. Immunol.
PUBLISHED: 02-22-2014
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Dicer is a multifunctional protein that is essential across species for the generation of microRNAs, a function that is highly conserved across the plant and animal kingdoms. Intriguingly, Dicer exhibits antiviral functions in lower organisms including Drosophila melanogaster and Caenorhabditis elegans. Antiviral activity occurs via small interfering RNA production following cytoplasmic sensing of viral dsRNA. Notably, such antiviral activity has not yet been clearly demonstrated in higher organisms such as mammals. Here, we review the evidence for Dicer as an innate antiviral across species.
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Influenza virus drug resistance: a time-sampled population genetics perspective.
PLoS Genet.
PUBLISHED: 02-01-2014
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The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.
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Role of human papillomavirus in cutaneous squamous cell carcinoma: a meta-analysis.
J. Am. Acad. Dermatol.
PUBLISHED: 01-06-2014
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The role of human papillomavirus (HPV) in cutaneous squamous cell carcinoma (cuSCC) is not well defined, with past studies showing conflicting results.
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Current management of fecal incontinence.
Perm J
PUBLISHED: 12-21-2013
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To review the management of fecal incontinence, which affects more than 1 in 10 people and can have a substantial negative impact on quality of life.
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Evolution of the Influenza A Virus Genome during Development of Oseltamivir Resistance In Vitro.
J. Virol.
PUBLISHED: 10-23-2013
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Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.
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Capture and printing of fixed stromal cell membranes for bioactive display on PDMS surfaces.
Langmuir
PUBLISHED: 08-12-2013
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Poly(dimethylsiloxane) (PDMS) has emerged as an extremely useful polymer for various biological applications. The conjugation of PDMS with bioactive molecules to create functional surfaces is feasible yet limited to a single-molecule display with imprecise localization of the molecules on PDMS. Here we report a robust technique that can transfer and print the membrane surface of glutaraldehyde-fixed stromal cells intact onto a PDMS substrate using an intermediate polyvinylalcohol (PVA) film as a transporter system. The cell-PVA film capturing the entirety of surface molecules can be peeled off and subsequently printed onto PDMS while maintaining the spatial display of the original cell surface molecules. Proof-of-concept studies are described using human bone marrow stromal cell membranes including a demonstration of the bioactivity of transferred membranes to capture and adhere hematopoietic cells. The presented process is applicable to virtually any adherent cell and can broaden the functional display of biomolecules on PDMS for biotechnology applications.
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Interpersonal and Personal Antecedents and Consequences of Peer Victimization Across Middle Childhood in Hong Kong.
J Youth Adolesc
PUBLISHED: 08-09-2013
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Although much is known about peer victimization, the majority of the longitudinal research in this area has been restricted to Western settings. The main objective of this study was to examine the interpersonal (rejection) and personal (withdrawal, aggression) antecedents and consequences of victimization for Chinese children living in Hong Kong. A sample of 1,058 children (501 boys; M age = 9.5 years) in Hong Kong was followed longitudinally from the 3rd and 4th grades to the 7th and 8th grades. Consistent with a transactional framework, rejection and withdrawal contributed to, as well as resulted from, victimization. Although victimization predicted later aggression, aggression was unrelated to later victimization. These findings closely replicate past research conducted in North America and European settings, and suggest considerable correspondence in the links between maladaptive child characteristics and victimization across Western and Hong Kong schools.
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Photocycle of Exiguobacterium sibiricum rhodopsin characterized by low-temperature trapping in the IR and time-resolved studies in the visible.
J Phys Chem B
PUBLISHED: 06-10-2013
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The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.
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Germ cell specification.
Adv. Exp. Med. Biol.
PUBLISHED: 06-08-2013
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The germline of Caenorhabditis elegans derives from a single founder cell, the germline blastomere P(4). P(4) is the product of four asymmetric cleavages that divide the zygote into distinct somatic and germline (P) lineages. P(4) inherits a specialized cytoplasm ("germ plasm") containing maternally encoded proteins and RNAs. The germ plasm has been hypothesized to specify germ cell fate, but the mechanisms involved remain unclear. Three processes stand out: (1) inhibition of mRNA transcription to prevent activation of somatic development, (2) translational regulation of the nanos homolog nos-2 and of other germ plasm mRNAs, and (3) establishment of a unique, partially repressive chromatin. Together, these processes ensure that the daughters of P(4), the primordial germ cells Z2 and Z3, gastrulate inside the embryo, associate with the somatic gonad, initiate the germline transcriptional program, and proliferate during larval development to generate ?2,000 germ cells by adulthood.
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Breaking the carboxyl rule: lysine 96 facilitates reprotonation of the Schiff base in the photocycle of a retinal protein from Exiguobacterium sibiricum.
J. Biol. Chem.
PUBLISHED: 05-21-2013
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A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ?-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.
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Preference-for-solitude and adjustment difficulties in early and late adolescence.
J Clin Child Adolesc Psychol
PUBLISHED: 05-17-2013
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Social withdrawal has been associated with adjustment difficulties across development. Although much is known about shyness, little is known about preference-for-solitude; even less is known about its relations with adjustment across different periods of adolescence. We examined whether preference-for-solitude might be differentially associated with adjustment difficulties in early and late adolescence. Self- and parent-reports of withdrawal motivations and adjustment were collected from 234 eighth graders (113 boys; M age = 13.43) and 204 twelfth graders (91 boys; M age = 17.25). Results from structural equation modeling demonstrated that above and beyond the effects of shyness, preference-for-solitude was more strongly associated with adjustment difficulties in 8th grade than in 12th grade. Preference-for-solitude was associated with greater anxiety/depression, emotion dysregulation, and lower self-esteem in 8th grade; these relations were not found in 12th grade. Although preference-for-solitude was associated with lower social competence in both 8th and 12th grades, this relation was significantly stronger in 8th grade than in 12th grade. Findings suggest preference-for-solitude has closer ties to maladjustment in early adolescence than in late adolescence. Interventions targeting preferred-solitary youth in early adolescence may be particularly fruitful.
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Food-related advertisements and food intake among adult men and women.
Appetite
PUBLISHED: 05-02-2013
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Television viewing may contribute to obesity via promotion of sedentary behavior and exposure to food-related commercials. However, the mechanisms by which food-related commercials promote food intake are not well understood. Therefore, the purpose of the present study was to examine the impact of television advertisements on food intake according to sex and transportability, or the tendency to become engrossed in what one is viewing. Eighty-three undergraduate students, free of disordered eating symptoms, were stratified by sex and randomly assigned to one of three conditions (food-related advertisements, neutral advertisements, or no advertisements). They were then identified as high or low in transportability according to a median split. A significant interaction was found between advertisement condition and transportability such that those high in transportability ate more in the food than other advertisement conditions. A second interaction was found between sex and transportability with women high in transportability eating more food than women low in transportability irrespective of advertisement condition. No significant main effects of advertisement condition, sex, or transportability were found. Results suggest the importance of studying the impact of individual difference variables on the relationship between food-related advertising and food intake.
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Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.
Biotechnol. Bioeng.
PUBLISHED: 02-15-2013
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Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (?3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.
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Increased mammalian lifespan and a segmental and tissue-specific slowing of aging after genetic reduction of mTOR expression.
Cell Rep
PUBLISHED: 02-14-2013
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We analyzed aging parameters using a mechanistic target of rapamycin (mTOR) hypomorphic mouse model. Mice with two hypomorphic (mTOR(?/?)) alleles are viable but express mTOR at approximately 25% of wild-type levels. These animals demonstrate reduced mTORC1 and mTORC2 activity and exhibit an approximately 20% increase in median survival. While mTOR(?/?) mice are smaller than wild-type mice, these animals do not demonstrate any alterations in normalized food intake, glucose homeostasis, or metabolic rate. Consistent with their increased lifespan, mTOR(?/?) mice exhibited a reduction in a number of aging tissue biomarkers. Functional assessment suggested that, as mTOR(?/?) mice age, they exhibit a marked functional preservation in many, but not all, organ systems. Thus, in a mammalian model, while reducing mTOR expression markedly increases overall lifespan, it affects the age-dependent decline in tissue and organ function in a segmental fashion.
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Sialylneolacto-N-tetraose c (LSTc)-bearing liposomal decoys capture influenza A virus.
J. Biol. Chem.
PUBLISHED: 01-28-2013
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Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.
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Association of two BRM promoter polymorphisms with head and neck squamous cell carcinoma risk.
Carcinogenesis
PUBLISHED: 01-15-2013
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The SWI/SNF chromatin remodeling complex is an important regulator of gene expression that has been linked to cancer development. Expression of Brahma (BRM), a critical catalytic subunit of SWI/SNF, is lost in a variety of solid tumors. Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) have been correlated with BRM loss and elevated cancer risk. The aim(s) of this study were to examine BRM expression in head and neck squamous cell carcinoma (HNSCC) and to correlate BRM polymorphisms with HNSCC risk. BRM expression studies were performed on eight HNSCC cell lines and 76 surgically resected tumor samples. A case-control study was conducted on 668 HNSCC patients (oral cavity, oropharynx, larynx and hypopharynx) and 700 healthy matched controls. BRM expression was lost in 25% of cell lines and 16% of tumors. The homozygous genotype of each polymorphism was significantly associated with increased HNSCC risk [BRM-741: adjusted odds ratio (aOR) 1.75, 95% CI 1.2-2.3, P < 0.001; BRM-1321: aOR 1.65, 95% CI 1.2-2.2, P < 0.001]. Individuals that were homozygous for both BRM polymorphisms had a more than 2-fold increase in the risk of HNSCC (aOR 2.23, 95% CI 1.5-3.4, P < 0.001). A particularly elevated risk was seen within the oropharynx, human papillomavirus-positive subgroup for carriers of both homozygous variants (aOR 3.09, 95% CI 1.5-6.8, P = 0.004). BRM promoter polymorphisms appear to act as susceptibility markers of HNSCC with potential utility in screening, prevention and treatment.
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The Drosophila BTB domain protein Jim Lovell has roles in multiple larval and adult behaviors.
PLoS ONE
PUBLISHED: 01-01-2013
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Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB (Bric-a-brac,Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In lov(47) , Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. lov(47) mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult lov(47) males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. lov(47) adults also show more defective negative gravitaxis than the previously isolated lov(91Y) mutant. In contrast, lov(66) produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We propose a negative regulatory role for the DNA deleted in lov(66) .
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Role of specific innate immune responses in herpes simplex virus infection of the central nervous system.
J. Virol.
PUBLISHED: 12-14-2011
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Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.
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Capillary size exclusion chromatography with picogram sensitivity for analysis of monoclonal antibodies purified from harvested cell culture fluid.
J Chromatogr A
PUBLISHED: 09-09-2011
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Size exclusion chromatography (SEC) is widely used in the characterization and quality control of therapeutic proteins to detect aggregates. Aggregation is a carefully monitored quality attribute from the earliest stages of clinical development owing to the possibility of eliciting an immunogenic response in the patient. During early stage molecule assessment for cell culture production, small-scale screening experiments are performed to permit rapid turn-around of results so as to not delay timelines. We report the development of a capillary SEC methodology for preliminary molecule assessment to support the evaluation of therapeutic candidates at an early stage of development. By making several key modifications to a commercially available liquid chromatography system, we demonstrate a platform process to perform capillary SEC with excellent precision, picogram sensitivity and good ruggedness. The limit of quantitation was determined to be approximately 15 pg; picogram sensitivity for SEC analysis of monoclonal antibodies had not been achieved prior to this work. In addition, we have developed a method to capture low levels of antibody (1 ?g/mL) from harvested cell culture fluid (HCCF) for capillary SEC analysis. Up to 40% recovery efficiency was achieved using this micro-recovery method on 3 mL HCCF samples. Using early stage cell culture transient transfection samples, which typically have much lower titers than stable cell line samples, we demonstrate a consistent method for analyzing aggregates in low protein concentration HCCF samples for molecule assessment and early stage candidate screening.
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LPLUNC1 modulates innate immune responses to Vibrio cholerae.
J. Infect. Dis.
PUBLISHED: 09-07-2011
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Recent studies demonstrate that long palate, lung, and nasal epithelium clone 1 protein (LPLUNC1) is involved in immune responses to Vibrio cholerae, and that variations in the LPLUNC1 promoter influence susceptibility to severe cholera in humans. However, no functional role for LPLUNC1 has been identified.
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When the seemingly innocuous "stings": racial microaggressions and their emotional consequences.
Pers Soc Psychol Bull
PUBLISHED: 09-01-2011
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Commonplace situations that are seemingly innocuous may nonetheless be emotionally harmful for racial minorities. In the current article the authors propose that despite their apparent insignificance, these situations can be harmful and experienced as subtle racism when they are believed to have occurred because of their race. In Study 1, Asian Americans reported greater negative emotion intensity when they believed that they encountered a situation because of their race, even after controlling for other potential social identity explanations. Study 2 replicated this finding and confirmed that the effect was significantly stronger among Asian Americans than among White participants. These findings clarify how perceptions of subtle racial discrimination that do not necessarily involve negative treatment may account for the "sting" of racial microaggressions, influencing the emotional well-being of racial minorities, even among Asian Americans, a group not often expected to experience racism.
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Contributions of the MyD88-dependent receptors IL-18R, IL-1R, and TLR9 to host defenses following pulmonary challenge with Cryptococcus neoformans.
PLoS ONE
PUBLISHED: 07-14-2011
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Signaling via the adapter protein, MyD88, is important in the host defense against Cryptococcus neoformans infection. While certain Toll-like receptors (TLRs) can enhance the clearance of Cryptococcus, the contributions of MyD88-dependent, TLR-independent pathways have not been fully investigated. We examined the roles of IL-1R and IL-18R in vivo by challenging C57BL/6 mice with a lethal strain of Cryptococcus. We found that the absence of IL-18R, but not IL-1R, causes a shift in the survival curve following pulmonary delivery of a virulent strain of C. neoformans (H99). Specifically, IL-18R-deficient mice have significantly shorter median survival times compared to wild-type mice following infection. Cytokine analysis of lung homogenates revealed that deficiency of IL-IR, IL-18R, or MyD88 is associated with diminished lung levels of IL-1?. In order to compare these findings with those related to TLR-deficiency, we studied the effects of TLR9-deficiency and found that deficiency of TLR9 also affects the survival curve of mice following challenge with C. neoformans. Yet the lungs from infected TLR9-deficient mice have robust levels of IL-1?. In summary, we found that multiple signaling components can contribute the MyD88-dependent host responses to cryptococcal infection in vivo and each drives distinct pulmonary responses.
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Inhibitory effects of calcitriol on the growth of MCF-7 breast cancer xenografts in nude mice: selective modulation of aromatase expression in vivo.
Horm Cancer
PUBLISHED: 06-21-2011
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Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active metabolite of vitamin D, exerts many anticancer effects in breast cancer (BCa) cells. We have previously shown using cell culture models that calcitriol acts as a selective aromatase modulator (SAM) and inhibits estrogen synthesis and signaling in BCa cells. We have now examined calcitriol effects in vivo on aromatase expression, estrogen signaling, and tumor growth when used alone and in combination with aromatase inhibitors (AIs). In immunocompromised mice bearing MCF-7 xenografts, increasing doses of calcitriol exhibited significant tumor inhibitory effects (~50% to 70% decrease in tumor volume). At the suboptimal doses tested, anastrozole and letrozole also caused significant tumor shrinkage when used individually. Although the combinations of calcitriol and the AIs caused a statistically significant increase in tumor inhibition in comparison to the single agents, the cooperative interaction between these agents appeared to be minimal at the doses tested. Calcitriol decreased aromatase expression in the xenograft tumors. Importantly, calcitriol also acted as a SAM in the mouse, decreasing aromatase expression in the mammary adipose tissue, while increasing it in bone marrow cells and not altering it in the ovaries and uteri. As a result, calcitriol significantly reduced estrogen levels in the xenograft tumors and surrounding breast adipose tissue. In addition, calcitriol inhibited estrogen signaling by decreasing tumor ER? levels. Changes in tumor gene expression revealed the suppressive effects of calcitriol on inflammatory and growth signaling pathways and demonstrated cooperative interactions between calcitriol and AIs to modulate gene expression. We hypothesize that cumulatively these calcitriol actions would contribute to a beneficial effect when calcitriol is combined with an AI in the treatment of BCa.
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Microbial and environmental effects on avian egg viability: do tropical mechanisms act in a temperate environment?
Ecology
PUBLISHED: 06-14-2011
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The viability of freshly laid avian eggs declines after several days of exposure to ambient temperatures above physiological zero, and declines occur faster in tropical than temperate ecosystems. Microbial infection during preincubation exposure has recently been shown as a second cause of egg viability decline in the tropics, but whether microbial processes influence the viability of wild bird eggs in temperate ecosystems is unknown. We determined the microbial load on eggshells, the incidence of microbial penetration of egg contents, and changes in the viability of wild bird eggs (Sialia mexicana, Tachycineta bicolor, Tachycineta thalassina) experimentally exposed to temperate-zone ambient conditions in situ in a mediterranean climate in northern California. Initial microbial loads on eggshells were generally low, although they were significantly higher on eggs laid in old boxes than in new boxes. Eggshell microbial loads did not increase with exposure to ambient conditions, were not reduced by twice-daily disinfection with alcohol, and were unaffected by parental incubation. The rate of microbial penetration into egg contents was low and unaffected by the duration of exposure. Nevertheless, egg viability declined very gradually and significantly with exposure duration, and the rate of decline differed among species. In contrast to studies performed in the tropics, we found little evidence that temperature or microbial mechanisms of egg viability decline were important at our temperate-zone site; neither temperatures above physiological zero nor alcohol disinfection was significantly related to hatching success. Delaying the onset of incubation until the penultimate or last egg of a clutch at our study site may maintain hatching synchrony without a large trade-off in egg viability. These results provide insight into the environmental mechanisms that may be responsible for large-scale latitudinal patterns in avian clutch size and hatching asynchrony.
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Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis.
J. Immunol.
PUBLISHED: 05-23-2011
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IL-17 and IL-23 are known to be absolutely central to psoriasis pathogenesis because drugs targeting either cytokine are highly effective treatments for this disease. The efficacy of these drugs has been attributed to blocking the function of IL-17-producing T cells and their IL-23-induced expansion. However, we demonstrate that mast cells and neutrophils, not T cells, are the predominant cell types that contain IL-17 in human skin. IL-17(+) mast cells and neutrophils are found at higher densities than IL-17(+) T cells in psoriasis lesions and frequently release IL-17 in the process of forming specialized structures called extracellular traps. Furthermore, we find that IL-23 and IL-1? can induce mast cell extracellular trap formation and degranulation of human mast cells. Release of IL-17 from innate immune cells may be central to the pathogenesis of psoriasis, representing a fundamental mechanism by which the IL-23-IL-17 axis mediates host defense and autoimmunity.
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Structure of Sir2Tm bound to a propionylated peptide.
Protein Sci.
PUBLISHED: 04-19-2011
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Lysine propionylation is a recently identified post-translational modification that has been observed in proteins such as p53 and histones and is thought to play a role similar to acetylation in modulating protein activity. Members of the sirtuin family of deacetylases have been shown to have depropionylation activity, although the way in which the sirtuin catalytic site accommodates the bulkier propionyl group is not clear. We have determined the 1.8 Å structure of a Thermotoga maritima sirtuin, Sir2Tm, bound to a propionylated peptide derived from p53. A comparison with the structure of Sir2Tm bound to an acetylated peptide shows that hydrophobic residues in the active site shift to accommodate the bulkier propionyl group. Isothermal titration calorimetry data show that Sir2Tm binds propionylated substrates more tightly than acetylated substrates, but kinetic assays reveal that the catalytic rate of Sir2Tm deacylation of propionyl-lysine is slightly reduced to acetyl-lysine. These results serve to broaden our understanding of the newly identified propionyl-lysine modification and the ability of sirtuins to depropionylate, as well as deacetylate, substrates.
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On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format.
Anal. Biochem.
PUBLISHED: 04-05-2011
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Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography-mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.
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Are positive emotions just as "positive" across cultures?
Emotion
PUBLISHED: 03-30-2011
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Whereas positive emotions and feeling unequivocally good may be at the heart of well-being among Westerners, positive emotions often carry negative associations within many Asian cultures. Based on a review of East-West cultural differences in dialectical emotions, or co-occurring positive and negative feelings, we predicted culture to influence the association between positive emotions and depression, but not the association between negative emotions and depression. As predicted, in a survey of over 600 European-, immigrant Asian-, and Asian American college students, positive emotions were associated with depression symptoms among European Americans and Asian Americans, but not immigrant Asians. Negative emotions were associated with depression symptoms among all three groups. We also found initial evidence that acculturation (i.e., nativity) may influence the role of positive emotions in depression: Asian Americans fell "in between" the two other groups. These findings suggest the importance of studying the role of culture in positive emotions and in positive psychology. The use of interventions based on promoting positive emotions in clinical psychology among Asian clients is briefly discussed.
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The combination of early and rapid type I IFN, IL-1?, and IL-1? production are essential mediators of RNA-like adjuvant driven CD4+ Th1 responses.
PLoS ONE
PUBLISHED: 03-21-2011
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There is a growing need for novel vaccine adjuvants that can provide safe and potent T-helper type 1 (Th1) activity. RNA-like immune response modifiers (IRMs) are candidate T-cell adjuvants that skew acquired immune responses towards a Th1 phenotype. We set out to delineate the essential signaling pathways by which the RNA-like IRMs, resiquimod (R-848) and polyinosinic:polycytidylic acid (poly I:C), augment CD4+ T-helper 1 (Th1) responses. Highly purified murine conventional dendritic cells (cDCs) and conventional CD4+ T-cells were co-cultured in allogeneic and MHC congenic mixed leukocyte reactions. The activation of CD4+ Th1 cells was examined utilizing cells from mice deficient in specific RNA-sensing pattern recognition receptors and signaling mediators. R-848 and poly I:C stimulation of Type I interferon production and signaling in cDCs was essential but not sufficient for driving CD4+ Th1 responses. The early and rapid production of IL-1? and IL-1? was equally critical for the optimal activation of Th1 CD4+ T-cells. R-848 activation of Toll-like receptor 7/MyD88-dependent signaling in cDCs led to a rapid upregulation of pro-IL-1? and pro-IL-1? production compared to poly I:C activation of MyD88-independent signaling pathways. The in vitro data show that CD4+ T-cell adjuvant activity of RNA-like IRMs is mediated by a critical combination of early and rapid Type I interferon, IL-1? and IL-1? production. These results provide important insights into the key signaling pathways responsible for RNA-like IRM CD4+ Th1 activation. A better understanding of the critical signaling pathways by which RNA-like IRMs stimulate CD4+ Th1 responses is relevant to the rational design of improved vaccine adjuvants.
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A nonredundant role for plasmacytoid dendritic cells in host defense against the human fungal pathogen Aspergillus fumigatus.
Cell Host Microbe
PUBLISHED: 03-14-2011
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While plasmacytoid dendritic cells (pDCs), a natural type I interferon (IFN)-producing cell type, are regarded as critical for innate immunity to viruses, their role in defense against fungal infections remains unknown. We examined the interactions of pDCs with hyphae of the invasive human fungal pathogen Aspergillus fumigatus. Human pDCs spread over hyphae and inhibited their growth. Antifungal activity was retained in pDC lysates, did not require direct fungal contact, and was partially reversed by zinc. Incubation with hyphae resulted in pDC cytotoxicity, partly due to fungal gliotoxin secretion. Following hyphal stimulation, pDCs released proinflammatory cytokines via a TLR9-independent mechanism. Pulmonary challenge of mice with A. fumigatus resulted in a substantial influx of pDCs into lungs, and pDC-depleted mice were hypersusceptible to invasive aspergillosis. These data demonstrate the antifungal activity of pDCs against A. fumigatus and establish their nonredundant role in host defenses against invasive aspergillosis in vivo.
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Microfluidics-based diagnostics of infectious diseases in the developing world.
Nat. Med.
PUBLISHED: 02-03-2011
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One of the great challenges in science and engineering today is to develop technologies to improve the health of people in the poorest regions of the world. Here we integrated new procedures for manufacturing, fluid handling and signal detection in microfluidics into a single, easy-to-use point-of-care (POC) assay that faithfully replicates all steps of ELISA, at a lower total material cost. We performed this mChip assay in Rwanda on hundreds of locally collected human samples. The chip had excellent performance in the diagnosis of HIV using only 1 ?l of unprocessed whole blood and an ability to simultaneously diagnose HIV and syphilis with sensitivities and specificities that rival those of reference benchtop assays. Unlike most current rapid tests, the mChip test does not require user interpretation of the signal. Overall, we demonstrate an integrated strategy for miniaturizing complex laboratory assays using microfluidics and nanoparticles to enable POC diagnostics and early detection of infectious diseases in remote settings.
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Gender-specific differences in pelvic organ function after proctectomy for inflammatory bowel disease.
Dis. Colon Rectum
PUBLISHED: 02-02-2011
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Significant concern exists regarding the effect of proctectomy on sexual function in patients with IBD. Little is known about gender-specific differences.
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Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans.
Science
PUBLISHED: 12-02-2010
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Asymmetric segregation of P granules during the first four divisions of the Caenorhabditis elegans embryo is a classic example of cytoplasmic partitioning of germline determinants. It is thought that asymmetric partitioning of P granule components during mitosis is essential to distinguish germline from soma. We have identified a mutant (pptr-1) in which P granules become unstable during mitosis and P granule proteins and RNAs are distributed equally to somatic and germline blastomeres. Despite symmetric partitioning of P granule components, pptr-1 mutants segregate a germline that uniquely expresses P granules during postembryonic development. pptr-1 mutants are fertile, except at high temperatures. Hence, asymmetric partitioning of maternal P granules is not essential to specify germ cell fate. Instead, it may serve to protect the nascent germline from stress.
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Removal and reconstitution of the carotenoid antenna of xanthorhodopsin.
J. Membr. Biol.
PUBLISHED: 09-09-2010
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Salinixanthin, a C(40)-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein-lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the ?-system of the carotenoid polyene chain and immobilized in a binding site near the ?-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer.
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Solid-state interactions of a drug substance and excipients and their impact on tablet dissolution: a thermal-mechanical facilitated process-induced transformation or PIT.
J Pharm Sci
PUBLISHED: 06-10-2010
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The polymorphic and/or pseudo-polymorphic phase transformation of Drug Z API, from Form I to Form II, occurs during the wet granulation step. It was observed that dissolution of the tablets slowed down under certain manufacturing conditions. Factors responsible for the slowdown in tablet dissolution were investigated in this study. Two levels of shear during premixing and two wet granulation drying temperatures were investigated by measuring the dissolution profiles of the tablets. The interaction between API and excipients was examined using differential scanning calorimetry and X-ray powder diffraction. When stearic acid was present with Form I as the starting material in the formulations, the dissolution slowdown was significant under the conditions of higher shear during premixing or higher drying temperature. However, there was little impact of lower shear premixing or lower drying temperature. When Form I was replaced with Form II, the slowdown in dissolution was mainly observed with higher drying temperature. The tablet dissolution slowdown was due to the interaction between Form II and stearic acid that facilitated the formation of Form I. The transformation back to the Form I material reported here may be classified as a thermal-mechanical facilitated PIT and represents a new subclass of the phenomena.
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Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound.
J. Neurosci.
PUBLISHED: 05-14-2010
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The accumulation of amyloid beta (Abeta) in Alzheimers disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.
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Pollution magnet: nano-magnetite for arsenic removal from drinking water.
Environ Geochem Health
PUBLISHED: 05-04-2010
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Arsenic contamination in groundwater is a severe global problem, most notably in Southeast Asia where millions suffer from acute and chronic arsenic poisoning. Removing arsenic from groundwater in impoverished rural or urban areas without electricity and with no manufacturing infrastructure remains a significant challenge. Magnetite nanocrystals have proven to be useful in arsenic remediation and could feasibly be synthesized by a thermal decomposition method that employs refluxing of FeOOH and oleic acid in 1-octadecene in a laboratory setup. To reduce the initial cost of production, $US 2600/kg, and make this nanomaterial widely available, we suggest that inexpensive and accessible "everyday" chemicals be used. Here we show that it is possible to create functional and high-quality nanocrystals using methods appropriate for manufacturing in diverse and minimal infrastructure, even those without electricity. We suggest that the transfer of this knowledge is best achieved using an open source concept.
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Complement receptor 1 expression on mouse erythrocytes mediates clearance of Streptococcus pneumoniae by immune adherence.
Infect. Immun.
PUBLISHED: 05-03-2010
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Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from "extracellular" bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1(+) mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1(+) mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.
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Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.
Tissue Eng Part A
PUBLISHED: 04-23-2010
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We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.
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Fatty acid and water-soluble polymer-based controlled release drug delivery system.
J Pharm Sci
PUBLISHED: 02-25-2010
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Sustained release capsule formulations based on three components, drug, water-soluble polymer, and water-insoluble fatty acid, were developed. Theophylline, acetaminophen, and glipizide, representing a wide spectrum of aqueous solubility, were used as model drugs. Povidone and hydroxypropyl cellulose were selected as water-soluble polymers. Stearic acid and lauric acid were selected as water-insoluble fatty acids. Fatty acid, polymer, and drug mixture was filled into size #0 gelatin capsules and heated for 2 h at 50 °C. The drug particles were trapped into molten fatty acid and released at a controlled rate through pores created by the water-soluble polymer when capsules were exposed to an aqueous dissolution medium. Manipulation of the formulation components enabled release rates of glipizide and theophylline capsules to be similar to commercial Glucotrol XL tablets and Theo-24 capsules, respectively. The capsules also exhibited satisfactory dissolution stability after exposure to 30 °C/60% relative humidity (RH) in open Petri dishes and to 40 °C/75% RH in closed high-density polyethylene bottles. A computational fluid dynamic-based model was developed to quantitatively describe the drug transport in the capsule matrix and the drug release process. The simulation results showed a diffusion-controlled release mechanism from these capsules.
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Low-temperature FTIR study of multiple K intermediates in the photocycles of bacteriorhodopsin and xanthorhodopsin.
J Phys Chem B
PUBLISHED: 02-09-2010
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Low-temperature FTIR spectroscopy of bacteriorhodopsin and xanthorhodopsin was used to elucidate the number of K-like bathochromic states, their sequence, and their contributions to the photoequilibrium mixtures created by illumination at 80-180 K. We conclude that in bacteriorhodopsin the photocycle includes three distinct K-like states in the sequence bR (hv)--> I* --> J --> K(0) --> K(E) --> L --> ..., and similarly in xanthorhodopsin. K(0) is the main fraction in the mixture at 77 K that is formed from J. K(0) becomes thermally unstable above approximately 50 K in both proteins. At 77 K, both J-to-K(0) and K(0)-to-K(E) transitions occur and, contrarily to long-standing belief, cryogenic trapping at 77 K does not produce a pure K state but a mixture of the two states, K(0) and K(E), with contributions from K(E) of approximately 15 and approximately 10% in the two retinal proteins, respectively. Raising the temperature leads to increasing conversion of K(0) to K(E), and the two states coexist (without contamination from non-K-like states) in the 80-140 K range in bacteriorhodopsin, and in the 80-190 K range in xanthorhodopsin. Temperature perturbation experiments in these regions of coexistence revealed that, in spite of the observation of apparently stable mixtures of K(0) and K(E), the two states are not in thermally controlled equilibrium. The K(0)-to-K(E) transition is unidirectional, and the partial transformation to K(E) is due to distributed kinetics, which governs the photocycle dynamics at temperatures below approximately 245 K (Dioumaev and Lanyi, Biochemistry 2008, 47, 11125-11133). From spectral deconvolution, we conclude that the K(E) state, which is increasingly present at higher temperatures, is the same intermediate that is detected by time-resolved FTIR prior to its decay, on a time scale of hundreds of nanoseconds at ambient temperature (Dioumaev and Braiman, J. Phys. Chem. B 1997, 101, 1655-1662), into the K(L) state. We were unable to trap the latter separately from K(E) at low temperature, due to the slow distributed kinetics and the increasingly faster overlapping formation of the L state. Formation of the two consecutive K-like states in both proteins is accompanied by distortion of two different weakly bound water molecules: one in K(0), the other in K(E). The first, well-documented in bacteriorhodopsin at 77 K where K(0) dominates, was assigned to water 401 in bacteriorhodopsin. The other water molecule, whose participation has not been described previously, is disturbed on the next step of the photocycle, in K(E), in both proteins. In bacteriorhodopsin, the most likely candidate is water 407. However, unlike bacteriorhodopsin, the crystal structure of xanthorhodopsin lacks homologous weakly bound water molecules.
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Neurologic dysfunction and male infertility in Drosophila porin mutants: a new model for mitochondrial dysfunction and disease.
J. Biol. Chem.
PUBLISHED: 01-28-2010
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Voltage-dependent anion channels (VDACs) are a family of small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. VDACs play an important role in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, and three distinct mammalian isoforms have been identified. Animal and cell culture experiments suggest that the various isoforms act in disparate roles such as apoptosis, synaptic plasticity, learning, muscle bioenergetics, and reproduction. In Drosophila melanogaster, porin is the ubiquitously expressed VDAC isoform. Through imprecise excision of a P element insertion in the porin locus, a series of hypomorphic alleles have been isolated, and analyses of flies homozygous for these mutant alleles reveal phenotypes remarkably reminiscent of mouse VDAC mutants. These include partial lethality, defects of mitochondrial respiration, abnormal muscle mitochondrial morphology, synaptic dysfunction, and male infertility, which are features often observed in human mitochondrial disorders. Furthermore, the observed synaptic dysfunction at the neuromuscular junction in porin mutants is associated with a paucity of mitochondria in presynaptic termini. The similarity of VDAC mutant phenotypes in the fly and mouse clearly indicate a fundamental conservation of VDAC function. The establishment and validation of a new in vivo model for VDAC function in Drosophila should provide a valuable tool for further genetic dissection of VDAC role(s) in mitochondrial biology and disease, and as a model of mitochondrial disorders potentially amenable to the development of treatment strategies.
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Effects of human OEC-derived cell transplants in rodent spinal cord contusion injury.
Brain Res.
PUBLISHED: 01-27-2010
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Numerous reports indicate that rodent olfactory ensheathing cells (OECs) assist in spinal cord repair and clinical trials have been undertaken using autologous transplantation of human olfactory ensheathing cells (hOECs) as a treatment for spinal cord injury. However, there are few studies investigating the efficacy of hOECs in animal models of spinal cord injury. In this study hOECs were derived from biopsies of human olfactory mucosa, purified by culture in a serum-free medium containing neurotrophin-3, genetically labelled with EGFP, and stored frozen. These hOEC-derived cells were thawed and transplanted into the spinal cord injury site 7 days after a moderate contusion injury of the spinal cord at thoracic level T10 in the athymic rat. Six weeks later the animals receiving the hOEC-derived transplants had greater functional improvement in their hindlimbs than controls, assessed using open field (BBB scale) and horizontal rung walking tests. Histological analysis demonstrated beneficial effects of hOEC-derived cell transplantation: reductions in the volume of the lesion and the cavities within the lesion. The transplanted cells were located at the periphery of the lesion where they integrated with GFAP-positive astrocytes resulting in a significant reduction of GFAP staining intensity adjacent to the lesion. Although their mechanism of action is unclear we conclude that hOEC-derived cell transplants improved functional recovery after transplantation into the contused spinal cord, probably by modulating inflammatory responses and reducing secondary damage to the cord.
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Modeling of pan coating processes: Prediction of tablet content uniformity and determination of critical process parameters.
J Pharm Sci
PUBLISHED: 01-22-2010
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We developed an engineering model for predicting the active pharmaceutical ingredient (API) content uniformity (CU) for a drug product in which the active is coated onto a core. The model is based on a two-zone mechanistic description of the spray coating process in a perforated coating pan. The relative standard deviation (RSD) of the API CU of the coated tablets was found to be inversely proportional to the square root of the total number of cycles between the spray zone and drying zone that the tablets undergo. The total number of cycles is a function of the number of tablets in the drying zone, the spray zone width, the tablet velocity, the tablet number density, and the total coating time. The sensitivity of the RSD to various critical coating process parameters, such as pan speed, pan load, spray zone width, as well as tablet size and shape was evaluated. Consequently, the critical coating process parameters needed to achieve the desired API CU were determined. Several active film coating experiments at 50, 200, and 400 kg using various pan coaters demonstrated that good correlation between the model predictions and the experimental results for the API CU was achieved.
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Lubrication in tablet formulations.
Eur J Pharm Biopharm
PUBLISHED: 01-03-2010
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Theoretical aspects and practical considerations of lubrication in tablet compression are reviewed in this paper. Properties of the materials that are often used as lubricants, such as magnesium stearate, in tablet dosage form are summarized. The manufacturing process factors that may affect tablet lubrication are discussed. As important as the lubricants in tablet formulations are, their presence can cause some changes to the tablet physical and chemical properties. Furthermore, a detailed review is provided on the methodologies used to characterize lubrication process during tablet compression with relevant process analytical technologies. Finally, the Quality-by-Design considerations for tablet formulation and process development in terms of lubrication are discussed.
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Microfluidic point-of-care diagnostics for resource-poor environments.
Conf Proc IEEE Eng Med Biol Soc
PUBLISHED: 12-08-2009
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Point-of-care (POC) diagnostics have tremendous potential to improve human health in remote and resource-poor settings. However, the design criteria for diagnostic tests appropriate in settings with limited infrastructure are unique and challenging. Here we present a custom optical reader which quantifies silver absorbance from heterogeneous immunoassays. The reader is simple, low-cost and suited for POC diagnostics.
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Identification of codon-specific serine to asparagine mistranslation in recombinant monoclonal antibodies by high-resolution mass spectrometry.
Anal. Chem.
PUBLISHED: 10-27-2009
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Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.
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Epstein-Barr virus (EBV)-encoded RNA: automated in-situ hybridization (ISH) compared with manual ISH and immunohistochemistry for detection of EBV in pediatric lymphoproliferative disorders.
Pediatr. Dev. Pathol.
PUBLISHED: 09-26-2009
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Detection of Epstein-Barr virus (EBV) may be achieved by various methods, including EBV-encoded RNA (EBER) in-situ hybridization (ISH) and immunohistochemistry (IHC) for latent membrane protein (LMP-1). We compared novel automated ISH and IHC techniques in pediatric lymphoproliferative disorders with results obtained by manual ISH. Thirty-seven pediatric cases previously studied by manual EBER ISH (including 18 EBER-positive, 15 EBER-negative, and 4 EBER-equivocal cases) were used for the study. Automated EBER ISH and automated LMP-1 IHC were performed using the BondMax autostainer and prediluted EBER probe and EBV cell surface 1 to 4 at 1:50 dilution, respectively. Results of each of the automated techniques for EBV detection were compared with results by manual EBER ISH. Compared with manual EBER ISH as the gold standard, automated ISH had a sensitivity and specificity of 94% and 69%, respectively, accuracy of 83%, positive predictive value (PPV) of 79%, and negative predictive value (NPV) of 90%. Automated IHC had a sensitivity of 44%, specificity of 93%, accuracy of 67%, PPV of 88%, and NPV of 59%. Automated ISH and IHC correlated significantly (P < 0.045). Automated ISH is useful for diagnosis of EBV-related pediatric neoplasms, being easy to perform and interpret and requiring only the technologists time to set up and having a high sensitivity and NPV The automated IHC protocol is of too low sensitivity for routine use, although results show high specificity and PPV.
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A valid and reliable measure of constipation-related quality of life.
Dis. Colon Rectum
PUBLISHED: 07-21-2009
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Few existing measures assess constipation-specific quality of life. This study sought to develop a valid and reliable quality-of-life measure for constipation.
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Treatment of transsphincteric anal fistulas: are fistula plugs an acceptable alternative?
Dis. Colon Rectum
PUBLISHED: 05-01-2009
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Treatments for transsphincteric, cryptoglandular anal fistulas pose risks for high recurrence rates and impaired anal continence. Anal fistula plugs have gained popularity because of reports of success without compromising the anal sphincter. To examine the benefit of the anal fistula plug, we compared its success rate with a standard treatment for transsphincteric fistulas with similar indications, the transanal mucosal advancement flap.
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Laser-evoked synaptic transmission in cultured hippocampal neurons expressing channelrhodopsin-2 delivered by adeno-associated virus.
J. Neurosci. Methods
PUBLISHED: 04-01-2009
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We present a method for studying synaptic transmission in mass cultures of dissociated hippocampal neurons based on patch clamp recording combined with laser stimulation of neurons expressing channelrhodopsin-2 (ChR2). Our goal was to use the high spatial resolution of laser illumination to come as close as possible to the ideal of identifying monosynaptically coupled pairs of neurons, which is conventionally done using microisland rather than mass cultures. Using recombinant adeno-associated virus (rAAV) to deliver the ChR2 gene, we focused on the time period between 14 and 20 days in vitro, during which expression levels are high, and spontaneous bursting activity has not yet started. Stimulation by wide-field illumination is sufficient to make the majority of ChR2-expressing neurons spike. Stimulation with a laser spot at least 10 microm in diameter also produces action potentials, but in a reduced fraction of neurons. We studied synaptic transmission by voltage-clamping a neuron with low expression of ChR2 and scanning a 40 microm laser spot at surrounding locations. Responses were observed to stimulation at a subset of locations in the culture, indicating spatial localization of stimulation. Pharmacological means were used to identify responses that were synaptic. Many responses were of smaller amplitude than those typically found in microisland cultures. We were unable to find an entirely reliable criterion for distinguishing between monosynaptic and polysynaptic responses. However, we propose that postsynaptic currents with small amplitudes, simple shapes, and latencies not much greater than 8 ms are reasonable candidates for monosynaptic interactions.
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Distinct patterns of dendritic cell cytokine release stimulated by fungal beta-glucans and toll-like receptor agonists.
Infect. Immun.
PUBLISHED: 03-09-2009
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beta-Glucans derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Yeast glucan particles (YGPs) are highly purified Saccharomyces cerevisiae cell walls composed of beta1,6-branched beta1,3-d-glucan and free of mannans. YGPs stimulated secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in wild-type murine bone marrow-derived myeloid dendritic cells (BMDCs) but did not stimulate interleukin-12p70 (IL-12p70) production. A purified soluble beta1,6-branched beta1,3-d-glucan, scleroglucan, also stimulated TNF-alpha in BMDCs. These two beta-glucans failed to stimulate TNF-alpha in Dectin-1 (beta-glucan receptor) knockout BMDCs. Costimulation of wild-type BMDCs with beta-glucans and specific Toll-like receptor (TLR) ligands resulted in greatly enhanced TNF-alpha production but decreased IL-12p70 production compared with TLR agonists alone. The upregulation of TNF-alpha and downregulation of IL-12p70 required Dectin-1, but not IL-10. Gamma interferon (IFN-gamma) priming did not overcome IL-12p70 reduction by beta-glucans. Similar patterns of cytokine regulation were observed in human monocyte-derived dendritic cells (DCs) costimulated with YGPs and the TLR4 ligand lipopolysaccharide. Finally, costimulation of BMDCs with YGPs and either the TLR9 ligand, CpG, or the TLR2/1 ligand, Pam(3)CSK(4), resulted in upregulated secretion of IL-1alpha and IL-10 and downregulated secretion of IL-1beta, IL-6, and IFN-gamma-inducible protein 10 but had no significant effects on IL-12p40, keratinocyte-derived chemokine, monocyte chemotactic protein 1, or macrophage inflammatory protein alpha, compared with the TLR ligand alone. Thus, beta-glucans have distinct effects on cytokine responses following DC stimulation with different TLR agonists. These patterns of response might contribute to the skewing of immune responses during mycotic infections and have implications for the design of immunomodulators and vaccines containing beta-glucans.
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Hospitalized ulcerative colitis patients have an elevated risk of thromboembolic events.
World J. Gastroenterol.
PUBLISHED: 02-28-2009
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To compare thromboembolism rates between hospitalized patients with a diagnosis of ulcerative colitis and other hospitalized patients at high risk for thromboembolism. To compare thromboembolism rates between patients with ulcerative colitis undergoing a colorectal operation and other patients undergoing colorectal operations.
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Antiviral responses: different roles for different tolls.
Immunity
PUBLISHED: 02-26-2009
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In this issue of Immunity, Town et al. (2009) show the interplay between Toll-like receptors (TLRs) and cytokines during West Nile virus infection and define a role for TLR-mediated production of interleukin-23 in immune cell homing and pathogenesis.
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The cell adhesion molecule "CAR" and sialic acid on human erythrocytes influence adenovirus in vivo biodistribution.
PLoS Pathog.
PUBLISHED: 01-02-2009
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Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad-erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.
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Regions on adenylyl cyclase VII required for selective regulation by the G13 pathway.
Mol. Pharmacol.
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Regulation of multiple adenylyl cyclases (AC) provides unique inputs to mediate the synthesis of cAMP, a ubiquitous second messenger that controls many aspects of cellular function. On stimulation by G(s), the activities of ACs can be further selectively modulated by other pathways to ensure precise control of intracellular cAMP responses to specific stimuli. Recently, we reported that one of the AC isoforms, AC7, is uniquely regulated by the G(13) pathway. To understand more fully the molecular mechanism of this regulation, we compared the regulation of AC7 with that of AC2 in bone marrow-derived macrophages devoid of AC7. Although both enzymes could fully restore regulation of cAMP by G??, activation of the G(13) pathway preferentially synergized with AC7. Exchange of domains between the two isoforms indicates that the C1b domain and the N-terminus of the C1a domain are important for directing selective regulation of AC7 by the G(13) pathway. A mutagenesis screen identified more specific regions of AC7 that differentially mediate its regulation by distinct pathways.
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Zymosan activates protein kinase A via adenylyl cyclase VII to modulate innate immune responses during inflammation.
Mol. Immunol.
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Pathogens use a variety of strategies to evade host immune defenses. A powerful way to suppress immune function is to increase intracellular concentrations of cAMP in host immune cells, which dampens inflammatory responses and prevents microbial killing. We found that the yeast cell wall extract, zymosan, is capable of increasing intracellular cAMP and activates the protein kinase A pathway in bone marrow derived macrophage (BMDM) cells from mice. This response is dependent on adenylyl cyclase type VII (AC7) and heterotrimeric G proteins, primarily G(12/13). Consequently, zymosan induced production of the inflammatory cytokine, TNF?, was much stronger in BMDMs from AC7 deficient mice compared to the response in wild type cells. In a model of zymosan induced peritonitis, mice deficient of AC7 in the myeloid lineage displayed prolonged inflammation. We propose that zymosan induced increases in cAMP and activation of PKA serve as a mechanism to dampen inflammatory responses in host cells, which consequently favors the survival of microbes. This would also help explain a well documented phenomenon, that the ability of zymosan to stimulate inflammatory cytokine responses via TLR2 appears to be weaker than other stimuli of TLR2.
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Development of capillary size exclusion chromatography for the analysis of monoclonal antibody fragments extracted from human vitreous humor.
J Chromatogr A
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Recombinant antigen-binding fragments (Fabs) are currently on the market and in development for the treatment of ophthalmologic indications. Recently, Quality by Design (QbD) initiatives have been implemented that emphasize understanding the relationship between quality attributes of the product and their impact on safety and efficacy. In particular, changes in product quality once the protein is administered to the patient are of particular interest. Knowledge of protein aggregation in vivo is of importance due to the possibility of antibody aggregates eliciting an immunogenic response in the patient. Presently, there are few analytical methods with adequate sensitivity to analyze Fab aggregates in human vitreous humor (HVH) because the Fab amount available for analysis is often quite low. Here, we report the development of a highly sensitive capillary size exclusion chromatography (SEC) methodology for Fab aggregate analysis in HVH. We demonstrate a process to perform capillary SEC to analyze Fabs with picogram sensitivity and an RSD of less than 8% for the relative peak area of high molecular weight species (HMWS). In addition, we have developed a Protein G affinity chromatography method to capture Fabs from HVH for capillary SEC analysis. Recovery efficiencies ranging from 86 to 99% were achieved using this recovery method with 300 ?L HVH samples containing Fab1. Finally, we demonstrate the applicability of the methodology by quantifying Fab aggregates in HVH, which can potentially be used for aggregate analysis of clinically relevant samples.
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Plasmacytoid dendritic cell interferon-? production to R-848 stimulation is decreased in male infants.
BMC Immunol.
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Sex differences in response to microbial infections, especially viral ones, may be associated with Toll-like receptor (TLR)-mediated responses by plasmacytoid dendritic cells (pDCs).
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.